Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics

ABSTRACT

The invention provides improved non-human vertebrates and non-vertebrate cells capable of expressing antibodies comprising human variable region sequences. The present invention is directed to the provision of long HCDR3s from non-human vertebrates and cells. The present invention is also directed to the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially expanded diversities are provided. The invention also provides for novel and potentially expanded diversity or diversity that is biased towards variable gene usage common to antibodies useful for treating and/or preventing certain diseases or conditions, such as infectious diseases. The invention also provides methods of generating antibodies using such vertebrates, as well as the antibodies per se, therapeutic compositions thereof and uses.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.14/220,074 filed Mar. 19, 2014, which is a continuation of internationalapplication PCT/GB2012/052298 filed Sep. 18, 2012, which claims priorityto GB1116122.1 filed Sep. 19, 2011, GB1116120.5 filed Sep. 19, 2011,GB1203257.9 filed Feb. 24, 2012, GBBab filed Mar. 15, 2012, GB1205702.2filed Mar. 29, 2012, GB1208749.0 filed May 18, 2012 and GB1211692.7filed Jul. 2, 2012, all of which are herein incorporated by reference.

The attached sequence listing titled 39080-18401_seq_listing.txt (size 2kb and created on Jul. 6, 2021), is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the provision of antibodies with longHCDR3 lengths. The present invention is also directed to the provisionof novel V, D and J pairings in immunoglobulin heavy and light chainloci. Novel, biased antibody diversities and potentially expandeddiversities are provided. The invention also provides for novel andpotentially expanded diversity or diversity that is biased towardsvariable gene usage common to antibodies useful for treating and/orpreventing certain diseases or conditions, such as infectious diseases.This ability to bias the antibody repertoire also provides methods ofsimplifying the production of antibody mixtures, such as polyclonalantibody therapeutics useful for the treatment and/or prevention ofinfectious diseases where a polyclonal approach to target multiplepathogen antigens is desirable. To this end, the present invention alsoprovides bispecific antibodies that are capable of binding to more thanone antigen (eg, multiple infectious antigens expressed by the samepathogen), thus providing advantages (such as manufacturing, dosing andadministration advantages) not possible with polyclonal antibodymixtures.

The present invention provides vertebrates and cells, such as transgenicmice or rats or transgenic mouse or rat cells. Furthermore, theinvention relates to methods of using the vertebrates to isolateantibodies or nucleotide sequences encoding antibodies. Antibodies,nucleotide sequences, pharmaceutical compositions and uses are alsoprovided by the invention.

BACKGROUND

The state of the art provides non-human vertebrates (eg, mice and rats)and cells comprising transgenic immunoglobulin loci, such locicomprising human variable (V), diversity (D) and/or joining (J)segments, and optionally human constant regions. Alternatively,endogenous constant regions of the host vertebrate (eg, mouse or ratconstant regions) are provided in the transgenic loci. Methods ofconstructing such transgenic vertebrates and use of these to generateantibodies and nucleic acids thereof following antigen immunisation areknown in the art, eg, see U.S. Pat. No. 7,501,552 (Medarex), U.S. Pat.No. 5,939,598 (Abgenix), U.S. Pat. No. 6,130,364 (Abgenix), WO02/066630(Regeneron), WO2011004192 (Genome Research Limited), WO2009076464,WO2009143472 and WO2010039900 (Ablexis), the disclosures of which areexplicitly incorporated herein. Such transgenic loci in the art includevarying amounts of the human V(D) J repertoire.

Existing transgenic immunoglobulin loci tend to be based on a singlehuman DNA source. The potential diversity of human antibody variableregions in non-human vertebrates bearing such transgenic loci is thusconfined by the repertoire used.

It would be desirable to provide for novel and potentially expandedrepertoire and diversity of human variable regions in transgenicimmunoglobulin loci and non-human vertebrates harbouring these, as wellas in antibodies produced following immunisation of such animals.

SUMMARY OF THE INVENTION

The present inventors have discovered, by way of construction oftransgenic non-human vertebrates, immunisation, antibody heavy chaincollection and deep bioinformatics analysis, how to rationally designfor VH domains, heavy chains and antibodies having long HCDR3s. Theseare useful for addressing antigens (such as infectious disease pathogenantigens, receptors and enzyme clefts) where a longer CDR betteraddresses the target.

The present inventors also realised the possibility of providingcombinations of V, D and J gene segments in new ways to providesynthetic gene segment combinations in immunoglobulin loci that are notfound in nature or in state-of-the-art loci. The inventors realised theimportance of this to provide for novel and potentially expandedrepertoire and diversity of human variable regions in transgenicimmunoglobulin loci and non-human vertebrates harbouring these, as wellas in antibodies produced following immunisation of such animals. In oneaspect, the inventors realised that it would be desirable to bias thenovel repertoire for the production of antibodies having improvedaffinity and/or biophysical characteristics, and/or wherein the range ofepitope specificities produced by means of such repertoire is novel,provides for antibodies to epitopes that have hitherto been intractableby prior transgenic immunoglobulin loci or difficult to address. Forexample, the inventors envisaged a specific application to bias thenovel repertoire for the production of antibodies useful in the therapyand/or prevention of infectious disease.

To this end, in a first configuration of the invention, there isprovided A non-human vertebrate (optionally a mouse or a rat) orvertebrate cell whose genome comprises:

(a) An immunoglobulin heavy chain locus comprising one or more human Vgene segments, one or more human D gene segments and one or more human Jgene segments upstream of a constant region; optionally wherein theheavy chain locus is according to any configuration of the inventiondescribed below; and(b) An immunoglobulin light chain locus comprising either(i) one or more human VH gene segments and one or more human J genesegments upstream of a constant region (optionally a rearranged VHJLCLor VHJλCL, wherein the CL is Cλ or Cκ); or(ii) one or more human VL gene segments, one or more human D genesegments and one or more human JH gene segments upstream of a constantregion (optionally a rearranged VLDJHCL or VλDJHCL, wherein the CL is Cλor Cκ); or(iii) one or more human VL gene segments selected from the groupconsisting of: a VλII gene family member, VλVII 4A, VλII 2.1, VλVII 4A,a Vλ1 gene family member, a VX3gene family member, IGLV1S2, Vλ3-cML70,Ialh2, Ialvl, Ia3h3, Kv325, a VκI gene family member, κI-15A (KL012),VκII family member, a VκIII family member, a VκI gene family member,κI-15A (KL012), VκII A2 (optionally the A2a allele), Vκ A27 (Humkv325)and a gene segment at least 80% identical thereto, and one or more humanJL gene segments upstream of a constant region;

Wherein the gene segments in the heavy chain locus are operably linkedto the constant region thereof, and the gene segments in the light chainlocus are operably linked to the constant region thereof, so that uponimmunisation the mouse is capable of producing an antibody comprisingheavy chains produced by recombination of the heavy chain locus andlight chains produced by recombination of the light chain locus.

In one aspect,

in (b)(i) the V gene segment repertoire of the light chain locuscomprises or consists of one or more VH gene segments selected from thegroup consisting of: a VHIII gene family member (optionally, a VHIIIa orVHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15),VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11,VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%identical thereto; orin (b)(iii) the light chain locus V gene segment repertoire consists ofone VL gene segment type (optionally and one or mutants thereof),wherein the VL gene segment is selected from said group of VL genesegments.

In a second configuration of the present invention, there is provided

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

(a) An immunoglobulin heavy chain locus comprising one or more human Vgene segments, one or more human D gene segments and one or more human Jgene segments upstream of a constant region; and(b) (i) An unrearranged immunoglobulin light chain locus comprising oneor more human VH gene segments and one or more human J gene segmentsupstream of a constant region, wherein each human VH gene segment is ahuman gene segment identical to (or mutant of) a human VH gene segmentused to produce a rearranged VDJ encoding a heavy chain variable regionof a human antibody from an antibody-expressing cell wherein saidantibody binds to an antigen of an infectious disease pathogen(optionally the variable regions of said antibody being identical to anantibody from a human individual suffering, susceptible to, or recoveredfrom, a disease or condition caused or mediated by an organismharbouring or secreting said antigen; or from a human individualharbouring said organism); or(ii) An immunoglobulin light chain locus comprising a rearranged VJregion or VDJ region upstream of a constant region, wherein thenucleotide sequence of the recombined region is identical to anucleotide sequence produced by the recombination of a human J genesegment and optionally a human D gene segment with a human VH genesegment that is identical to (or mutant of) the human VH gene segmentused to produce a rearranged VDJ encoding a heavy chain variable regionof a human antibody from an antibody-expressing cell wherein saidantibody binds to an antigen of an infectious disease pathogen(optionally the variable regions of said antibody being identical to anantibody from a human individual suffering, susceptible to, or recoveredfrom, a disease or condition caused or mediated by an organismharbouring or secreting said antigen; or from a human individualharbouring said organism);(c) Wherein the gene segments in the heavy chain locus are operablylinked to the constant region thereof, and the gene segments or VJ orVDJ in the light chain locus are operably linked to the constant regionthereof, so that upon immunisation the mouse is capable of producing anantibody comprising heavy chains produced by recombination of the heavychain locus and light chains derived from the light chain locus;(d) Optionally when (b)(i) applies, each said VH gene segment in thelight chain locus is selected from the group consisting of: a VHIII genefamily member (optionally, a VHIIIa or VHIIIb family member), a VHIVgene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21,LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto;(e) Optionally when (b)(ii) applies, the nucleotide sequence of therecombined region is identical to a nucleotide sequence produced by therecombination of a human J gene segment and optionally a human D genesegment with a human VH gene segment selected from the group consistingof: a VHIII gene family member (optionally, a VHIIIa or VHIIIb familymember), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26(VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2,VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11,71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto.

In one aspect, the V gene segment repertoire of the light chain locuscomprises or consists of one human VH gene segment; optionally germlineVH and one or more polymorphic variants thereof, eg, where eachpolymorphic variant differs from the germline VH nucleotide sequence by1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions. In one aspect, the V genesegment repertoire of the light chain locus comprises or consists ofhuman VH1-69 gene segment; optionally germline VH1-69 and one or morepolymorphic variants thereof, eg, where each polymorphic variant differsfrom the germline VH1-69 nucleotide sequence by 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 positions. An example of constructing an immunoglobulin locuscomprising VH1-69 and polymorphic variants is given below. By using aparticular gene segment (eg, one commonly found in human antibodiesraised in humans against an infection or other condition) and one ormore polymorphic variants thereof, it is possible to provide arepertoire of genes and yet still bias the antibody gene repertoire to agene segment that is relevant to the disease (eg, an infectious disease,such as a bacterial or viral disease, eg, influenza). This provides auseful pool of genes from which to ultimately generate and isolate alead antibody for a therapeutic/prophylactic against the disease inquestion. In an example, the polymorphic variants are natural variantsseen in human beings or human populations. The skilled person will knowof sources of human antibody gene sequences, such as IMGT(www.imgt.org), GenBank (www.ncbi.nlm.nih.gov/genbank) and the 1000Genomes databases (www.1000genomes.org). Bioinformatics tools fordatabase manipulation are also readily available and known to theskilled person, eg, as publicly available from the 1000 GenomesProject/EBI (www.1000genomes.org)

In another aspect, the genome of said vertebrate or cell is homozygousfor light chain locus (b)(i) or (ii); optionally wherein:

the V gene segment repertoire of the light chain loci consists of one ormore human VH gene segments selected from the group consisting of: aVHIII gene family member (optionally, a VHIIIa or VHIIIb family member),a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto; or

-   -   the recombined VJ or VDJ repertoire of the light chain loci        consists of sequences identical to one or more nucleotide        sequences produced by the recombination of a human VH gene        segment selected from the group consisting of: a VHIII gene        family member (optionally, a VHIIIa or VHIIIb family member), a        VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26        (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR,        ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,        Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least        80% identical thereto, with a human J gene segment and        optionally a human D gene segment.

In another aspect, each immunoglobulin light chain locus of saidvertebrate or cell is according to (b)(i) and comprises only a singlehuman VH gene segment selected from the group consisting of: a VHIIIgene family member (optionally, a VHIIIa or VHIIIb family member), aVHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto, optionallywherein the genome of the vertebrate or cell is homozygous for saidlight chain so that all light chain loci comprise the same, single humanVH gene segment.

The invention provides a first method of isolating an antibody thatbinds a predetermined antigen, the method comprising

(a) providing a vertebrate (optionally a mouse or rat) according to anypreceding configuration or aspect;(b) immunising said vertebrate with said antigen (optionally wherein theantigen is an antigen of an infectious disease pathogen);(c) removing B lymphocytes from the vertebrate and selecting one or moreB lymphocytes expressing antibodies that bind to the antigen;(d) optionally immortalising said selected B lymphocytes or progenythereof, optionally by producing hybridomas therefrom; and(e) isolating an antibody (eg, and IgG-type antibody) expressed by the Blymphocytes.

A second method is provided comprising carrying out the first method andthe step of isolating from said B lymphocytes nucleic acid encoding saidantibody that binds said antigen; optionally exchanging the heavy chainconstant region nucleotide sequence of the antibody with a nucleotidesequence encoding a human or humanised heavy chain constant region andoptionally affinity maturing the variable region of said antibody; andoptionally inserting said nucleic acid into an expression vector andoptionally a host.

An aspect provides method of producing a polyclonal antibody mixture,the method comprising carrying out the first method by separatelyimmunising first and second vertebrates (optionally first and secondmice or first and second rats) with antigen and combining theanti-antigen antibodies isolated from each vertebrate (or mutants orderivatives of said antibodies) to produce a polyclonal antibodymixture; optionally wherein the following apply separately or incombination ((i) and (ii); or (i) and (iii)):

(i) the vertebrates are immunised with the same antigen or differentantigens (optionally wherein the different antigens are expressed by thesame pathogenic organism (or a family member thereof));(ii) prior to immunisation the light chain loci of the vertebratescontain the identical VH gene repertoire (optionally a single VH gene)and optionally the identical J repertoire; optionally the light chainloci of the mammals are identical prior to immunisation;(iii) prior to immunisation the light chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the light chain loci of the vertebratesare identical prior to immunisation.

An aspect provides method of producing a polyclonal antibody mixture,the method comprising carrying out the first method by immunising one ora plurality of vertebrates (optionally mice or rats) with first andsecond antigens and combining the anti-antigen antibodies isolated fromeach vertebrate (or mutants or derivatives of said antibodies) toproduce a polyclonal antibody mixture; optionally wherein the followingapply separately or in combination ((i) and (ii); or (i) and (iii)):

(i) the antigens are expressed by the same pathogenic organism (or afamily member thereof));(ii) prior to immunisation the light chain loci of the vertebratescontain the identical VH gene repertoire (optionally a single VH gene)and optionally the identical J repertoire; optionally the light chainloci of the mammals are identical prior to immunisation;(iii) prior to immunisation the light chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the light chain loci of the vertebratesare identical prior to immunisation.

An aspect provides method of producing host cells capable of expressinga polyclonal antibody mixture, the method comprising, in the secondmethod:—

(a) immunising one or a plurality of vertebrates (optionally mice orrats) with first and second antigens (optionally wherein the differentantigens are expressed by the same pathogenic organism (or a familymember thereof));(b) isolating nucleic acid encoding first and second anti-antigenantibodies from B lymphocytes from said vertebrates;(c) determining the nucleotide sequences of the heavy and light chainvariable regions of the first antibody;(d) determining the nucleotide sequence of the heavy variable region andoptionally the light chain variable region of the second antibody;(e) inserting the heavy chain variable region coding sequence of eachantibody into a heavy chain expression vector; optionally wherein theconstant region coding sequence of each heavy chain is exchanged for anucleotide sequence that encodes a human or humanised constant region;(f) inserting the light chain variable region coding sequence of thefirst antibody into a light chain expression vector; optionally whereinthe constant region coding sequence of the light chain of the firstantibody is exchanged for a nucleotide sequence that encodes a human orhumanised constant region;(g) optionally inserting the light chain variable region coding sequenceof the second antibody into a light chain expression vector; optionallywherein the constant region coding sequence of the light chain of thesecond antibody is exchanged for a nucleotide sequence that encodes ahuman or humanised constant region; and(h) introducing each expression vector into a host cell andco-expressing antibody chains in a mixture of said host cells to produceantibodies, each antibody comprising one or both of said heavy chainvariable regions and a light chain; optionally wherein the expressionvectors are introduced together into the same host cell (eg, a CHO orHEK293 cell) so that the cell is capable of expressing antibody lightchains and heavy chains, such that the cell or a plurality of the hostcells express antibodies, each comprising one or both of said heavychain variable regions and a light chain;(i) optionally:prior to immunisation the light chain loci of the vertebrates containthe identical VH gene repertoire (optionally a single VH gene segment)and optionally the identical J repertoire (optionally a single J genesegment); optionally the light chain loci of the vertebrates areidentical prior to immunisation; orprior to immunisation the light chain loci of the vertebrates containthe identical rearranged VJ or VDJ repertoire (optionally a single VJ orVDJ); optionally the light chain loci of the vertebrates are identicalprior to immunisation.

The invention also provides a method of producing a monoclonal orpolyclonal antibody mixture, optionally for use in medicine, optionallyfor the treatment and/or prevention of an infectious disease, whereinoptionally wherein each antibody binds an antigen of an infectiousdisease pathogen, preferably the same antigen. The invention alsoprovides the use of an isolated, monoclonal or polyclonal antibody, or amutant or derivative antibody thereof in the manufacture of a medicamentfor the treatment and/or prevention of an infectious disease, optionallywherein the infectious disease is a disease caused by a bacterial orviral pathogen.

The invention further provides an isolated antibody (eg, IgG-typeantibody) obtainable or obtained by a method of the invention, or amutant or derivative antibody thereof wherein (i) the isolated antibodycomprises two copies of the heavy chain variable region of said firstantibody paired with two copies of the light chain variable region ofsaid first antibody; or (ii) the isolated antibody comprises two copiesof the heavy chain variable region of said second antibody paired withtwo copies of the light chain variable region of said first antibody; or(iii) the isolated antibody is a bispecific antibody comprising one copyof the heavy chain variable region of said first antibody paired with acopy of the light chain variable region of the first antibody, and onecopy of the heavy chain variable region of said the antibody paired witha copy of the light chain variable region of the first antibody,optionally wherein the bispecific antibody binds to said first andsecond antigens are expressed by the same pathogenic organism (or afamily member thereof; optionally for use in medicine, optionally forthe treatment and/or prevention of an infectious disease.

In an aspect, there is provided a nucleotide sequence encoding anantibody of the invention, optionally wherein the nucleotide sequence ispart of a vector.

In an aspect, there is provided a pharmaceutical composition comprisingthe antibody or antibodies of the invention and a diluent, excipient orcarrier.

In a third configuration of the invention, there is provided A non-humanvertebrate (optionally a mouse or a rat) or vertebrate cell whose genomecomprises:

(a) An immunoglobulin heavy chain locus comprising either:—(i) one or more human VL gene segments, one or more human D genesegments and one or more human J gene segments upstream of a constantregion (optionally a rearranged VLDJHCH or VλDJHCH); or(ii) one or more human VH gene segments selected from the groupconsisting of: a VHIII gene family member (optionally, a VHIIIa orVHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15),VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11,VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%identical; one or more human D gene segments and one or more human JHgene segments upstream of a constant region; and(b) An immunoglobulin light chain locus comprising one or more human Vgene segments and one or more human J gene segments upstream of aconstant region, optionally wherein the light chain locus is accordingto (b)(i) or (b)(ii) of the first configuration of the invention;

Wherein the gene segments in the heavy chain locus are operably linkedto the constant region thereof, and the gene segments in the light chainlocus are operably linked to the constant region thereof, so that uponimmunisation the mouse is capable of producing an antibody comprisingheavy chains produced by recombination of the heavy chain locus andlight chains produced by recombination of the light chain locus.

In a fourth configuration of the invention, there is provided

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

(a) (i) An unrearranged immunoglobulin heavy chain locus comprising oneor more human VL gene segments, one or more human D gene segments andone or more JH gene segments upstream of a constant region, wherein eachhuman VL gene segment is a human gene segment identical to (or mutantof) a human VL gene segment used to produce a rearranged VJ encoding alight chain variable region of a human antibody from anantibody-expressing cell wherein said antibody binds to an antigen of aninfectious disease pathogen (optionally the variable regions of saidantibody being identical to an antibody from a human individualsuffering, susceptible to, or recovered from, a disease or conditioncaused or mediated by an organism harbouring or secreting said antigen;or from a human individual harbouring said organism); or(ii) An immunoglobulin heavy chain locus comprising a rearranged VJregion or VDJ region upstream of a constant region, wherein thenucleotide sequence of the recombined region is identical to anucleotide sequence produced by the recombination of a human J genesegment and optionally a human D gene segment with a human VL genesegment that is identical to (or mutant of) the human VL gene segmentused to produce a rearranged VJ encoding a light chain variable regionof a human antibody from an antibody-expressing cell wherein saidantibody binds to an antigen of an infectious disease pathogen(optionally the variable regions of said antibody being identical to anantibody from a human individual suffering, susceptible to, or recoveredfrom, a disease or condition caused or mediated by an organismharbouring or secreting said antigen; or from a human individualharbouring said organism);(b) An immunoglobulin light chain locus comprising one or more human Vgene segments and one or more human J gene segments upstream of aconstant region; and(c) Wherein the gene segments in the light chain locus are operablylinked to the constant region thereof, and the gene segments or VJ orVDJ in the heavy chain locus are operably linked to the constant regionthereof, so that upon immunisation the mouse is capable of producing anantibody comprising light chains produced by recombination of the lightchain locus and heavy chains derived from the heavy chain locus;(d) Optionally when (a)(i) applies, each said VL gene segment in theheavy chain locus is selected from the group consisting of a VL genesegment selected from the group consisting of a VλII gene family member,VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene family member, a Vλ3gene familymember, IGLV1S2, Vλ3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a VκI genefamily member, κI-15A (KL012), VκII family member, a VκIII familymember, a VκI gene family member, κI-15A (KL012), VκII A2 (optionallythe A2a allele), Vκ A27 (Humkv325) and a gene segment at least 80%identical thereto;(e) Optionally when (a)(ii) applies, the nucleotide sequence of therecombined region is identical to a nucleotide sequence produced by therecombination of a human J gene segment and optionally a human D genesegment with a human VL gene segment selected from the group consistingof a VL gene segment selected from the group consisting of a VλII genefamily member, VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene family member, aVλ3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, aVκI gene family member, κI-15A (KL012), VκII family member, a VκIIIfamily member, a VκI gene family member, κI-15A (KL012), VκII A2(optionally the A2a allele), VK A27 (Humkv325) and a gene segment atleast 80% identical thereto.

In one aspect of the fourth configuration of the invention, the genomeof said vertebrate or cell is homozygous for heavy chain locus (a)(i) or(ii); optionally wherein:

the V gene segment repertoire of the heavy chain loci consists of one ormore human VL gene segments selected from the group consisting of a VLgene segment selected from the group consisting of a VλII gene familymember, VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene family member, aVX3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, aVκI gene family member, κI-15A (KL012), VκII family member, a VκIIIfamily member, a VκI gene family member, κI-15A (KL012), VκII A2(optionally the A2a allele), VK A27 (Humkv325) and a gene segment atleast 80% identical thereto; or

the recombined VJ or VDJ repertoire of the heavy chain loci consists ofsequences identical to one or more nucleotide sequences produced by therecombination of a human VL gene segment selected from the groupconsisting of a VL gene segment selected from the group consisting of aVλII gene family member, VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene familymember, a VX3gene family member, IGLV1S2, VX3-cML70, Ialh2, Ialvl,Ia3h3, Kv325, a VκI gene family member, κI-15A (KL012), VκII familymember, a VκIII family member, a VκI gene family member, κI-15A (KL012),VκII A2 (optionally the A2a allele), Vκ A27 (Humkv325) and a genesegment at least 80% identical thereto with a human J gene segment andoptionally a human D gene segment.

The invention provides a monoclonal or polyclonal antibody compositionprepared by immunisation of at least one vertebrate (eg, mouse or rat)according to any preceding configuration or aspect with an antigen,optionally wherein the antigen is an antigen of an infectious diseasepathogen, optionally wherein the same antigen is used to immunise allthe vertebrates; optionally wherein the antibody or antibodies areIgG-type.

The invention also provides an isolated chimaeric antibody for treatingand/or preventing an infectious disease or condition, the antibodycomprising a non-human vertebrate (optionally a mouse or rat) heavychain constant regions and human variable regions that bind an antigenof an infectious disease pathogen, wherein the antibody is obtainable orobtained in a method comprising immunisation of a non-human vertebrateof the invention with said antigen.

The invention also provides an isolated human antibody for treatingand/or preventing an infectious disease or condition, the antibodycomprising human heavy chain constant regions and human variable regionsthat bind an antigen of an infectious disease pathogen, wherein theantibody is obtainable or obtained in a method comprising affinitymaturation of antibody variable regions in vivo in a transgenicnon-human vertebrate (eg, mouse or rat) when said variable regions areoperably linked to heavy chain constant regions of said vertebrate (eg,mouse or rat heavy chain constant regions) by (a) immunisation of avertebrate of the invention with said antigen, (b) isolation of nucleicacid encoding a chimaeric antibody according to the invention, (c)replacing the nucleotide sequences of the nucleic acid that encode thenon-human vertebrate heavy chain constant regions with nucleotidesequence encoding human heavy chain constant regions to produce nucleicacid encoding a human antibody; (d) expressing the human antibody invitro (optionally from CHO or HEK293 cells harbouring the human nucleicacid) and (e) isolating the human antibody (optionally with furtheraffinity maturation of the antibody and/or producing a derivativethereof).

An aspect provides a mixture of first and second human antibodies, eachantibody being capable of binding to an antigen of an infectious diseasepathogen (optionally wherein the first antibody binds a first antigenand the second antibody binds a second antigen, said antigens being fromthe same pathogen; or wherein the antigens are the same). In anembodiment, a common light chain is used which enables simplifiedmanufacture of the antibody mixture. Thus, there is provided in themixture, the light chain amino acid sequence of the first antibody thatis identical to the light chain amino acid sequence of the secondantibody, or has up to 15 amino acid changes therefrom.

The invention further provides a host cell comprising one or moreexpression vectors encoding 3 or more first and second antibody heavyand light chains.

In a fifth configuration of the invention, there is provided A syntheticimmunoglobulin locus comprising one or more variable and J gene segments(and optionally one or more D gene segments) operably linked 5′ of aconstant region, wherein the locus comprises a 5′ to 3′ V(D)Jarrangement selected from the group consisting of immunoglobulin locuscan be constructed with one or more of the following arrangements (5′ to3′):—

-   (a) [V (heavy, lambda or kappa)]—[two-turn RSS]—[one-turn    RSS]—[D]—[JH], wherein said RSSs are in an opposite orientation;-   (b) [VH]—[D]—[two-turn RSS]—[one-turn RSS]—[J lambda], wherein said    RSSs are in an opposite orientation;-   (c) [VH]—[D]—[one-turn RSS]—[two-turn RSS]—[J kappa], wherein said    RSSs are in an opposite orientation;-   (d) [VH or V kappa]-[two-turn RSS]—[one-turn RSS]—[J lambda],    wherein said RSSs are in an opposite orientation;-   (e) [V kappa]-[one-turn RSS]—[two-turn RSS]—[JH or J lambda],    wherein said RSSs are in an opposite orientation;-   (f) [V (heavy, lambda or kappa)]—[one-turn RSS]—[two-turn    RSS]—[D]—[JH], wherein said RSSs are in an opposite orientation;-   (g) [VH]—[D]—[one-turn RSS]—[two-turn RSS]—[J lambda], wherein said    RSSs are in an opposite orientation;-   (h) [VH]—[D]—[two-turn RSS]—[one-turn RSS]—[J kappa], wherein said    RSSs are in an opposite orientation;-   (i) [VH or V kappa]-[one-turn RSS]—[two-turn RSS]—[J lambda],    wherein said RSSs are in an opposite orientation;-   (j) [V kappa]-[two-turn RSS]—[one-turn RSS]—[JH or J lambda],    wherein said RSSs are in an opposite orientation.

In a sixth configuration, the invention also provides means forgenerating VH domains, heavy chains and antibodies having a long HCDR3length. In this context, the invention provides:—

A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebratecell (eg, a mouse cell or a rat cell) whose genome comprises a humanimmunoglobulin D gene segment repertoire that is biased to the human D2and/or D3 family or biased to one, more or all human D gene segmentsselected from the group D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13and D6-19.

A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebratecell (eg, a mouse cell or a rat cell), optionally according to anypreceding claim, whose genome comprises a human immunoglobulin VH genesegment repertoire that is biased to one, more or all of gene segmentsselected from the group VH1-2, VH1-3, VH1-8, VH1-18, VH5-51, VH1-69,VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4,VH6-1 and VH7-4-1.

A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebratecell (eg, a mouse cell or a rat cell) whose genome comprises a humanimmunoglobulin JH gene segment repertoire that is biased to human JH6.

A monoclonal or polyclonal antibody composition or a population ofantibody-producing cells for producing such composition, wherein thecomposition or population is prepared by immunising at least onevertebrate according to any preceding claim with an antigen, wherein theantibody or antibodies have human heavy chain variable regionscomprising non-human vertebrate AID-pattern somatic hypermutations, (eg,mouse or rat AID-pattern mutations) when compared to corresponding humangermline V, D and J sequences and/or non-human (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern junctional mutationswhen compared to corresponding human germline V, D and J sequences;wherein the composition comprises at least one antigen-specific antibodyhaving a HCDR3 length of at least 20 amino acids (according to IMGT).

A repertoire of antibody heavy chains (eg, provided by antibodies)comprising one or more heavy chains whose variable domain HCDR3 has alength of at least 20 amino acids (according to IMGT) and derived fromthe recombination of a human VH, D and JH, wherein the VH is selectedfrom the group

VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01,VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 andVH7-4-1*01 and

the D is selected from the group

D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or D2-2*02, D3-9*01 andD3-10*01, or D3-9*01 and D3-10*01, or D1-26, D2-2, D3-9, D3-10, D3-22,D4-17, D6-13 and D6-19, or D1-26*01, D2-2*02, D3-9*01, D3-10*01,D3-22*01, D4-17*01, D6-13*01 and D6-19*01, or D2-2, D3-9, D3-10, D3-22,D4-17, D6-13 and D6-19, or D2-2*02, D3-9*01, D3-10*01, D3-22*01,D4-17*01, D6-13*01 and D6-19*01, or D1-26, D2-2, D3-10 and D6-19, orD2-2, D3-9 and D3-10;

and optionally the JH is JH6 (eg, JH6*02);

Wherein

(a) the heavy chain variable domain has been produced in vivo in anon-human vertebrate (eg, a mouse or a rat); and/or(b) the heavy chain variable domain comprises non-human vertebrateAID-pattern somatic hypermutations, (eg, mouse or rat AID-patternmutations) when compared to corresponding human germline V, D and Jsequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyltransferase (TdT)-pattern junctional mutations when compared tocorresponding human germline V, D and J sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3: Schematic illustrating a protocol for producingrecombineered BAC vectors to add V gene segments into a mouse genome;

FIG. 4: Schematic illustrating a protocol for adding V gene segments toa mouse genome using sequential recombinase mediated cassette exchange(sRMCE);

FIG. 5 (in 4 parts): Alignment of 13 IGHV1-69 alleles showing thevariable (V) coding region only. Nucleotides that differ from VH1-69allele *01 are indicated at the appropriate position whereas identicalnucleotides are marked with a dash. Where nucleotide changes result inamino acid differences, the encoded amino acid is shown above thecorresponding triplet. Boxed regions correspond to CDR1, CDR2 and CDR3as indicated; and

FIG. 6: RSS structure and recombination schematic.

DETAILED DESCRIPTION OF THE INVENTION

A source for human V, D and J gene segments is Bacterial ArtificialChromosomes (RPCI-11 BACs) obtained from Roswell Park Cancer Institute(RPCI)/Invitrogen. See http://bacpac.chori.org/hmale11.htm, whichdescribes the BACs as follows:—

“RPCI—11 Human Male BAC Library

The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001) wasconstructed using improved cloning techniques (Osoegawa et al., 1998)developed by Kazutoyo Osoegawa. The library was generated by KazutoyoOsoegawa. Construction was funded by a grant from the National HumanGenome Research Institute (NHGRI, NIH) (#1R01RG01165-03). This librarywas generated according to the new NHGRI/DOE “Guidance on Human Subjectsin Large-Scale DNA Sequencing . . . .

“Male blood was obtained via a double-blind selection protocol. Maleblood DNA was isolated from one randomly chosen donor (out of 10 maledonors)”.

-   -   Osoegawa K, Mammoser A G, Wu C, Frengen E, Zeng C, Catanese J J,        de Jong P J; Genome Res. 2001 March; 11(3):483-96; “A bacterial        artificial chromosome library for sequencing the complete human        genome”;    -   Osoegawa, K., Woon, P. Y., Zhao, B., Frengen, E., Tateno, M.,        Catanese, J. J, and de Jong, P. J. (1998); “An Improved Approach        for Construction of Bacterial Artificial Chromosome Libraries”;        Genomics 52, 1-8.

As a source of antibody gene segment sequences, the skilled person willalso be aware of the following available databases and resources(including updates thereof):—

1.1. The Kabat Database (G. Johnson and T. T. Wu, 2002;http://www.kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in1966, the Kabat database publishes aligned sequences of antibodies,T-cell receptors, major histocompatibility complex (MHC) class I and IImolecules, and other proteins of immunological interest. A searchableinterface is provided by the SeqhuntII tool, and a range of utilities isavailable for sequence alignment, sequence subgroup classification, andthe generation of variability plots. See also Kabat, E. A., Wu, T. T.,Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteinsof Immunological Interest, 5th ed., NIH Publication No. 91-3242,Bethesda, Md., which is incorporated herein by reference, in particularwith reference to human gene segments for use in the present invention.1.2. KabatMan (A. C. R. Martin, 2002;http://www.bioinf.org.uk/abs/simkab.html). This is a web interface tomake simple queries to the Kabat sequence database.1.3. IMGT, the International ImMunoGeneTics Information System®; M. -P.Lefranc, 2002; http://imqt.cines.fr). IMGT is an integrated informationsystem that specializes in antibodies, T cell receptors, and MHCmolecules of all vertebrate species. It provides a common portal tostandardized data that include nucleotide and protein sequences,oligonucleotide primers, gene maps, genetic polymorphisms,specificities, and two-dimensional (2D) and three-dimensional (3D)structures. IMGT includes three sequence databases (IMGT/LIGM-DB,IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3Dstructure database (IMGT/3Dstructure-DB), and a range of web resources(“IMGT Marie-Paule page”) and interactive tools.1.4. V-BASE (I. M. Tomlinson, 2002; http://www.mrc-cpe.cam.ac.uk/vbase).V-BASE is a comprehensive directory of all human antibody germlinevariable region sequences compiled from more than one thousand publishedsequences. It includes a version of the alignment software DNAPLOT(developed by Hans-Helmar Althaus and Werner Müller) that allows theassignment of rearranged antibody V genes to their closest germline genesegments.1.5. Antibodies—Structure and Sequence (A. C. R. Martin, 2002;http://www.bioinf.org.uk/abs). This page summarizes useful informationon antibody structure and sequence. It provides a query interface to theKabat antibody sequence data, general information on antibodies, crystalstructures, and links to other antibody-related information. It alsodistributes an automated summary of all antibody structures deposited inthe Protein Databank (PDB). Of particular interest is a thoroughdescription and comparison of the various numbering schemes for antibodyvariable regions.1.6. AAAAA—AHo's Amazing Atlas of Antibody Anatomy (A. Honegger, 2001;http://www.unizh.ch/˜antibody). This resource includes tools forstructural analysis, modeling, and engineering. It adopts a unifyingscheme for comprehensive structural alignment of antibody andT-cell-receptor sequences, and includes Excel macros for antibodyanalysis and graphical representation.1.7. WAM—Web Antibody Modeling (N. Whitelegg and A. R. Rees, 2001;http://antibody.bath.ac.uk). Hosted by the Centre for Protein Analysisand Design at the University of Bath, United Kingdom. Based on the AbMpackage (formerly marketed by Oxford Molecular) to construct 3D modelsof antibody Fv sequences using a combination of established theoreticalmethods, this site also includes the latest antibody structuralinformation.1.8. Mike's Immunoglobulin Structure/Function Page (M. R. Clark, 2001;http://www.path.cam.ac.uk/˜mrc7/mikeimages.html) These pages provideeducational materials on immunoglobulin structure and function, and areillustrated by many colour images, models, and animations. Additionalinformation is available on antibody humanization and Mike Clark'sTherapeutic Antibody Human Homology Project, which aims to correlateclinical efficacy and anti-immunoglobulin responses with variable regionsequences of therapeutic antibodies.1.9. The Antibody Resource Page (The Antibody Resource Page, 2000;http://www.antibodyresource.com). This site describes itself as the“complete guide to antibody research and suppliers.” Links to amino acidsequencing tools, nucleotide antibody sequencing tools, andhybridoma/cell-culture databases are provided.1.9. Humanization bY Design (J. Saldanha, 2000;http://people.cryst.bbk.ac.uk/˜ubcq07s). This resource provides anoverview on antibody humanization technology. The most useful feature isa searchable database (by sequence and text) of more than 40 publishedhumanized antibodies including information on design issues, frameworkchoice, framework back-mutations, and binding affinity of the humanizedconstructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo,Methods in Molecular Biology™, Human Press. Also athttp://www.blogsua.com/pdf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

In one embodiment throughout the present text, “germline” refers to thecanonical germline gene segment sequence.

The present invention is directed to the provision of novel V, D and Jpairings in immunoglobulin heavy and light chain loci. Novel, biasedantibody diversities and potentially expanded diversities are provided.One aspect of the invention exploits the natural pairing of compatiblerecombination signal sequences (RSSs) during antibody V(D)Jrecombination in vivo, and this aspect of the invention provides new,synthetic combinations of V, D and J gene segments using the observationof RSS compatibility.

Another aspect of the invention is based on the observation of V, D andJ usage bias in naturally-occurring human antibodies raised againstinfectious disease pathogens. The invention is useful for manipulatingthe antibody gene diversity in transgenic non-human animals, thusproviding for novel and potentially expanded diversity or diversity thatis biased towards variable gene usage common to antibodies useful fortreating and/or preventing certain diseases or conditions, such asinfectious diseases. This ability to bias the antibody repertoire alsoprovides methods of simplifying the production of antibody mixtures,such as polyclonal antibody therapeutics useful for the treatment and/orprevention of infectious diseases where a polyclonal approach to targetmultiple pathogen antigens is desirable. To this end, the presentinvention also provides bispecific antibodies that are capable ofbinding to more than one antigen (eg, multiple infectious antigensexpressed by the same pathogen), thus providing advantages (such asmanufacturing, dosing and administration advantages) not possible withpolyclonal antibody mixtures.

The present invention provides vertebrates and cells, such as transgenicmice or rats or transgenic mouse or rat cells. Furthermore, theinvention relates to methods of using the vertebrates to isolateantibodies or nucleotide sequences encoding antibodies. Antibodies,nucleotide sequences, pharmaceutical compositions and uses are alsoprovided by the invention.

To this End, the Present Invention Provides, in a First Configuration

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

-   (a) An immunoglobulin heavy chain locus comprising one or more human    V gene segments (optionally a plurality of VH), one or more human D    gene segments and one or more human J gene segments upstream of a    constant region; optionally wherein the heavy chain locus is    according to (a) of the second configuration described below; and-   (b) An immunoglobulin light chain locus comprising either    -   (i) one or more human VH gene segments and one or more human J        gene segments upstream of a constant region (optionally a        rearranged V_(H)J_(L)C_(L) or V_(H)J_(A)C_(L), wherein the C_(L)        is C_(λ) or C_(K)); or    -   (ii) one or more human VL gene segments, one or more human D        gene segments and one or more human J_(H) gene segments upstream        of a constant region (optionally a rearranged V_(L)DJ_(H)C_(L)        or V_(λ)DJ_(H)C_(L), wherein the C_(L) is C_(λ) or C_(K)); or    -   (iii) one or more human VL gene segments selected from the group        consisting of: a V_(λ)II gene family member, V_(λ)VII 4A,        V_(λ)II 2.1, V_(λ)VII 4A, a V_(λ)1 gene family member, a V_(λ)3        gene family member, IGLV1S2, V_(λ)3-cML70, Ialh2, Ialvl, Ia3h3,        Kv325, a VκI gene family member, κI-15A (KL012), V_(κ)II family        member, a V_(κ)III family member, a VκI gene family member,        κI-15A (KL012), V_(κ)II A2 (optionally the Ata allele), V_(κ)        A27 (Humkv325) and a gene segment at least 80% identical        thereto, and one or more human J_(L) gene segments upstream of a        constant region; optionally the one or more VL gene segments are        selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4        below.    -   Wherein the gene segments in the heavy chain locus are operably        linked to the constant region thereof, and the gene segments in        the light chain locus are operably linked to the constant region        thereof, so that upon immunisation the mouse is capable of        producing an antibody comprising heavy chains produced by        recombination of the heavy chain locus and light chains produced        by recombination of the light chain locus.

This configuration of the invention, thus, provides for the possibilityof novel, synthetic antibody and gene repertoires in a transgenicnon-human vertebrate, such as a mouse or rat. Such new repertoires aredesirable, since they provide for the possibility of a novel pool ofantibodies from which lead antibodies can be selected followingimmunisation of the vertebrate with a predetermined antigen. This,therefore, provides for a pool from which antibodies with desirablecharacteristics can be isolated, for example, antibodies with relativelyhigh affinity for specific target antigen binding. It is desirable toisolate high affinity antibodies directly from the immunised vertebrate,since this can provide for an antibody lead that is potentially usefulas a therapeutic and/or prophylactic medicament without the need forfurther extensive affinity maturation (eg, by in vitro antibody displaysuch as ribosome display or phage display). Modification of the effectorportions of the antibody can be made as desired (eg, humanisation of theconstant region), without the need to manipulate the sequences of thevariable regions. Alternatively, or additionally, the pool of antibodiesmay allow for selection of a lead antibody with desirable biophysicalcharacteristics and/or epitope specificity. The latter may be importantfor finding lead antibodies against epitopes that have not previouslyraised therapeutic and/or prophylactic antibodies or epitopes that aredifficult to reach by antibodies generated by antibody gene diversitiesgenerated by prior non-human vertebrates bearing transgenicimmunoglobulin loci, eg, those based on the single human genomerepresented by the RPCI-11 BACs.

The cells of the invention (according to any aspect or configuration)is, for example, a B-cell, hybridoma or a stem cell, optionally anembryonic stem cell or haematopoietic stem cell. In one aspect the EScell is derived from the mouse C57BL/6N, C57BL/6J, 129S5 or 129Svstrain. In one aspect the non-human vertebrate is a rodent, suitably amouse, and cells of the invention, are rodent cells or ES cells,suitably mouse ES cells. The ES cells of the present invention can beused to generate animals using techniques well known in the art, whichcomprise injection of the ES cell into a blastocyst followed byimplantation of chimaeric blastocystys into females to produce offspringwhich can be bred and selected for homozygous recombinants having therequired insertion. In one aspect the invention relates to a transgenicanimal comprised of ES cell-derived tissue and host embryo derivedtissue. In one aspect the invention relates to genetically-alteredsubsequent generation animals, which include animals having a homozygousrecombinants for the VDJ and/or VJ regions.

Vertebrates bearing one or more light chain loci according to (b)(i) and(ii) provide for novel and potentially expanded antibody and generepertoires by exploiting synthetic, non-naturally-occurring,combinations of immunoglobulin gene segments (V, D, J, C). In thisrespect, the present inventors have realised the desirability andpossibility of providing for antibody and gene repertoires that mixheavy chain gene segments with those of light chain loci. This is basedon observations of the inventors: Firstly, nature suggests thepossibility of functional antibodies having VH-VH or VL-VL pairings (asopposed to more classical VH-VL pairings). For example, reference ismade to heavy chain antibodies of Camelidae which produce antibodieswith paired VH domains and is devoid of light chain VL domains (eg, seeNature. 1993 Jun. 3; 363(6428):446-8; Naturally occurring antibodiesdevoid of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S,Robinson G, Hamers C, Songa E B, Bendahman N, Hamers R). Theseantibodies function to specifically bind antigen, such antibodies beingfound in the blood of such Camelidae (eg, llamas, camels, alpacas). Suchantibodies with VH pairs can also be synthetically produced to providetherapeutic and prophylactic medicaments (eg, see WO1994004678,WO2004041862, WO2004041863). Transgenic mice also can produce such heavychain antibodies and the in vivo production of the antibodies allows themouse's immune system to select for VH-VH pairings, sometimes selectingfor such pairings in which mutations have been introduced in vivo by themouse to accommodate the pairing (WO2010109165A2). Thus, the inventorsrealised that the adoption of an in vivo antibody production system(rather than an in vitro system such as phage or ribosome display ofantibodies) is desirable to accommodate the synthetic immunoglobulingene segment combinations that are now contemplated by the presentinvention.

A second observation of the present inventors lies in the architectureof naturally-occurring immunoglobulin loci, and in particular thearrangement of recombination signal sequences (RSSs) that mediate V(D)Jrecombination in vivo (see, eg, Cell. 2002 April; 109 Suppl:S45-55. Themechanism and regulation of chromosomal V(D)J recombination; Bassing CH, Swat W, Alt F W, the disclosure of which is incorporated herein byreference). As illustrated in FIG. 6, two types of RSS element have beenidentified: a one-turn RSS (12-RSS) and a two-turn RSS (23-RSS). Innatural VJ recombination in the lambda light chain locus, recombinationif effected between a two-turn RSS that lies 3′ of a V lambda and aone-turn RSS that lies 5′ of a J lambda, the RSSs being in oppositeorientation. In natural VJ recombination in the kappa light chain locus,recombination if effected between a one-turn RSS that lies 3′ of a Vkappa and a two-turn RSS that lies 5′ of a J kappa, the RSSs being inopposite orientation. In natural VD recombination in the heavy chainlocus, recombination if effected between a two-turn RSS that lies 3′ ofa VH and a one-turn RSS that lies 5′ of a D, the RSSs being in oppositeorientation. In natural DJ recombination in the heavy chain locus,recombination if effected between a one-turn RSS that lies 3′ of a D anda two-turn RSS that lies 5′ of a JH, the RSSs being in oppositeorientation. Thus, generally a two-turn RSS is compatible with aone-turn RSS in the opposite orientation. The inventors realised thatthey could use this observation in constructing transgenicimmunoglobulin loci such that a 5′ gene segment can recombine with a 3′gene segment (eg, a V with a J; or a V with a D) when there is provideda two-turn RSS and a one-turn RSS in the opposite orientation, with eachRSS adjacent a respective one of the gene segments. Thus, the inventorsrealised in one embodiment that an immunoglobulin locus can beconstructed with one or more of the following arrangements (5′ to 3′):—

-   (k) [V (heavy, lambda or kappa)]—[two-turn RSS]—[one-turn    RSS]—[D]—[JH], wherein said RSSs are in an opposite orientation;-   (l) [VH]—[D]—[two-turn RSS]—[one-turn RSS]—[J lambda], wherein said    RSSs are in an opposite orientation;-   (m) [VH]—[D]—[one-turn RSS]—[two-turn RSS]—[J kappa], wherein said    RSSs are in an opposite orientation;-   (n) [VH or V kappa]-[two-turn RSS]—[one-turn RSS]—[J lambda],    wherein said RSSs are in an opposite orientation;-   (o) [V kappa]-[one-turn RSS]—[two-turn RSS]—[JH or J lambda],    wherein said RSSs are in an opposite orientation;-   (p) [V (heavy, lambda or kappa)]—[one-turn RSS]—[two-turn    RSS]—[D]—[JH], wherein said RSSs are in an opposite orientation;-   (q) [VH]—[D]—[one-turn RSS]—[two-turn RSS]—[J lambda], wherein said    RSSs are in an opposite orientation;-   (r) [VH]—[D]—[two-turn RSS]—[one-turn RSS]—[J kappa], wherein said    RSSs are in an opposite orientation;-   (s) [VH or V kappa]-[one-turn RSS]—[two-turn RSS]—[J lambda],    wherein said RSSs are in an opposite orientation;-   (t) [V kappa]-[two-turn RSS]—[one-turn RSS]—[JH or J lambda],    wherein said RSSs are in an opposite orientation.

The skilled person will realise that standard molecular biologytechniques can be used to provide vectors comprising syntheticcombinations of RSS with V, D or J for use in this aspect of theinvention, such that the vectors can be used to build a transgenicimmunoglobulin locus (eg, using homologous recombination and/orrecombinase mediated cassette exchange as known in the art, eg, see U.S.Pat. No. 7,501,552 (Medarex), U.S. Pat. No. 5,939,598 (Abgenix), U.S.Pat. No. 6,130,364 (Abgenix), WO02/066630 (Regeneron), WO2011004192(Genome Research Limited), WO2009076464, WO2009143472 and WO2010039900(Ablexis), the disclosures of which are explicitly incorporated herein.For example, such synthetic combinations with RSS and gene segments canbe made using standard recombineering techniques in E. coli to constructBAC vectors harbouring the synthetic combination prior to insertion inembryonic stem cells using homologous recombination or RMCE (eg, usingcre/lox site-specific recombination). Details of recombineering can befound at www.genebridges.com and in EP1034260 and EP1204740 thedisclosures of which are explicitly incorporated herein.

In one embodiment of (b)(i), all of the light chain locus V genesegments are human VH gene segments (optionally with one or more human Vlambda gene segments).

In one embodiment of (b)(i), the constant region is a mouse, rat orhuman CL, eg, Cλ. In one embodiment, the J and constant regions areprovided by one or more human JλCλ.

Although having utility generally to any antigen and disease setting,vertebrates bearing one or more light chain loci according to (b)(iii)are useful, in particular, for generating antibody leads againstinfectious disease pathogens. In this respect, the present inventorshave realised the desirability and possibility of providing for antibodyand gene repertoires that are biased to immunoglobulin gene segmentscommonly found in natural antibody reactions of humans to infectiousdisease pathogens. The inventors realised that it would be desirable toprovide for vertebrates, cells, methods etc for the production oftherapeutic and/or prophylactic antibodies based on natural human immuneresponses to antigens, such as antigens of infectious disease pathogens.In this respect, the literature observes frequently used immunoglobulingene segments to raise anti-infective responses in humans (Table 1).

TABLE 1 Immunoglobulin Gene Usage in Human Antibody Responses toInfectious Disease Pathogens REFERENCES V GENE ANTIGEN ORGANISM [HumanAb Source] BACTERIAL PATHOGENS KAPPA V GENES Haemophilus influenzae typeb Haemophilus influenzae 1. Lonberg, Nat Biotech 2005; Vk II germlinegene A2 + JK3 polysaccharide (Hib PS) [human PBMCs] Vk II family gene +JK4 2. Adderson et al, J Clin Invest 1992; 94% identical to the A27[Human PBLs] (Humkv325) germ line gene 3. Chung et al, J Immunol 1993 aVκI gene family member; κI- 4. Nadel et al, J Immunol 1998 15A (KL012)5. Feeney et al, J Clin Invest 1996 LAMBDA V GENES 6. Lucas et al,Infect Immun 1994; Four Vλ VII family members that [Human PBLs] are96-98% identical to 7. Adderson et al, J Clin Invest 1993; each other[Human PBLs] Vλ II family members (82, 89 and 8. Granoff et al, J ClinInvest 1993; 91% homologous to Vλ2.1 gene) [Human PBLs] +VHIII segmentsclosely 9. Azmi et al, Infect Immun 1994; homologous to germline tonsilcells gene 9.1 V_(λ)VII 4A All with Jλ homologous to germline Jλ2 andJλ3 VH GENES VH 96% identical to the VH germ line gene segment DP77(V3-21) LSG6.1, LSG12.1, V_(H)III VH26, V_(H)III 9.1 VH and VLCOMBINATIONS V_(H)III 9.1 + V_(λ)VII 4A V_(H)III 9.1 + V_(λ)II 2.1V_(H)III 9.1 + V_(κ)II A2 V_(H)III VH26 + V_(λ)II 2.1 V_(H)III 9.1;V_(H)III H11; V_(H)III VH26 Polysaccharide capsule E coli K1 9. Azmi etal, Infect Immun 1994 of E coli κI 15A K1 Vλ2.1 Meningococcal Bpolysaccharide; Neisseria meningitidis Group BPoly[α(2→8)-N-acetylneuramic acid VIRAL PATHOGENS VHIII or VHIV familymember Herpes family virus 10. Huang et al, J Clin Invest 1992; VλI orVλ3 member HSV 120-kD glycoprotein Herpes simplex virus (HSV); [humantonsils] HSV-1; HSV-2 VH26 + Dk1 + JH6 with IGLV1S2 + 116-, 105-, 64-kDglycoproteins Varicella zoster virus (VZV) Jλ2 of VZV VH4.18 VH2-1(VH3) + D region Dxp'1 + JH5 with Vλ3 cML70 + Jλ3 VH1GRR + JH3 + Dn4r orD2r with IGLV1S2 + Jλ2 For VZV Abs: ha3h2 (VH3) with IaIh2 (Vλ); orha1c1 (VH1) with IaIvI (Vλ1) For VZV Abs: ha4h3 (VH4) with Ia3h3 (Vλ3)Hv1051 (VH) Cytomegalovirus (CMV) 10. Huang et al, J Clin Invest 1992;Kv325 (Vk) 71-2 (VH) HIV 10. Huang et al, J Clin Invest 1992; Hv1f10(VH) 11. Wang & Palese, Science 2011 VH4.11 71-4 (VH) VH251 VH1-69VH1-69 Haemagglutinin (HA) Influenza virus, eg, Group 1 12. Ekiert etal, Science 2009 and/or Group 2 Infulenza A 13. Throsby et al, PLoS One2008 virus; eg, H1N1, H2N2, or 14. Sui et al, Nat Struct Mol Biol 2009H3N2 or H7N2 or H7N7 15. Ekiert et al, Science 2011 influenza virus

REFERENCES

-   1. Nat Biotechnol. 2005 September; 23(9):1117-25; Human antibodies    from transgenic animals; Lonberg N.-   2. J Clin Invest. 1992 March; 89(3):729-38; Immunoglobulin light    chain variable region gene sequences for human antibodies to    Haemophilus influenzae type b capsular polysaccharide are dominated    by a limited number of V kappa and V lambda segments and VJ    combinations; Adderson E E, Shackelford P G, Insel R A, Quinn A,    Wilson P M, Carroll W L.-   3. J Immunol. 1993 Oct. 15; 151(8):4352-61; Clonal characterization    of the human IgG antibody repertoire to Haemophilus influenzae type    b polysaccharide. V. In vivo expression of individual antibody    clones is dependent on Ig CH haplotypes and the categories of    antigen; Chung G H, Scott M G, Kim K H, Kearney J, Siber G R,    Ambrosino D M, Nahm M H.-   4. J Immunol. 1998 Dec. 1; 161(11):6068-73; Decreased frequency of    rearrangement due to the synergistic effect of nucleotide changes in    the heptamer and nonamer of the recombination signal sequence of the    V kappa gene Alb, which is associated with increased susceptibility    of Navajos to Haemophilus influenzae type b disease; Nadel B, Tang    A, Lugo G, Love V, Escuro G, Feeney A J.-   5. J Clin Invest. 1996 May 15; 97(10):2277-82; A defective Vkappa A2    allele in Navajos which may play a role in increased susceptibility    to Haemophilus influenzae type b disease; Feeney A J, Atkinson M J,    Cowan M J, Escuro G, Lugo G.-   6. Infect Immun. 1994 September; 62(9):3873-80; Variable region    sequences of a protective human monoclonal antibody specific for the    Haemophilus influenzae type b capsular polysaccharide; Lucas A H,    Larrick J W, Reason D C.-   7. J Clin Invest. 1993 June; 91(6):2734-43; Restricted    immunoglobulin VH usage and VDJ combinations in the human response    to Haemophilus influenzae type b capsular polysaccharide. Nucleotide    sequences of monospecific anti-Haemophilus antibodies and    polyspecific antibodies cross-reacting with self antigens; Adderson    E E, Shackelford P G, Quinn A, Wilson P M, Cunningham M W, Insel R    A, Carroll W L.-   8. J Clin Invest. 1993 March; 91(3):788-96; Variable region    expression in the antibody responses of infants vaccinated with    Haemophilus influenzae type b polysaccharide-protein conjugates.    Description of a new lambda light chain-associated idiotype and the    relation between idiotype expression, avidity, and vaccine    formulation. The Collaborative Vaccine Study Group; Granoff D M,    Shackelford P G, Holmes S J, Lucas A H.-   9. Infect Immun. 1994 May; 62(5):1776-86; Variable region sequences    and idiotypic expression of a protective human immunoglobulin M    antibody to capsular polysaccharides of Neisseria meningitidis group    B and Escherichia coli K1; Azmi F H, Lucas A H, Raff H V, Granoff D    M.-   10. J Clin Invest. 1992 December; 90(6):2197-208; Sequence analyses    of three immunoglobulin G anti-virus antibodies reveal their    utilization of autoantibody-related immunoglobulin Vh genes, but not    V lambda genes; Huang D F, Olee T, Masuho Y, Matsumoto Y, Carson D    A, Chen P P.-   11. Science. 2011 Aug. 12; 333(6044):834-5, Biochemistry. Catching a    moving target, Wang T T, Palese P-   12. Science. 2009 Apr. 10; 324(5924):246-51. Epub 2009 Feb. 26;    Antibody recognition of a highly conserved influenza virus epitope;    Ekiert D C, Bhabha G, Elsliger M A, Friesen R H, Jongeneelen M,    Throsby M, Goudsmit J, Wilson I A.-   13. PLoS One. 2008; 3(12):e3942. Epub 2008 Dec. 16; Heterosubtypic    neutralizing monoclonal antibodies cross-protective against H5N1 and    H1N1 recovered from human IgM+ memory B cells; Throsby M, van den    Brink E, Jongeneelen M, Poon L L, Alard P, Cornelissen L, Bakker A,    Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I,    Carsetti R, Peiris M, de Kruif J, Goudsmit J.-   14. Nat Struct Mol Biol. 2009 March; 16(3):265-73. Epub 2009 Feb.    22, Structural and functional bases for broad-spectrum    neutralization of avian and human influenza A viruses, Sui J, Hwang    W C, Perez S, Wei G, Aird D, Chen L M, Santelli E, Stec B, Cadwell    G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox N J, Bankston L    A, Donis R O, Liddington R C, Marasco W A.-   15. Science. 2011 Aug. 12; 333(6044):843-50. Epub 2011 Jul. 7, A    highly conserved neutralizing epitope on group 2 influenza A    viruses, Ekiert D C, Friesen R H, Bhabha G, Kwaks T, Jongeneelen M,    Yu W, Ophorst C, Cox F, Korse H J, Brandenburg B, Vogels R,    Brakenhoff J P, Kompier R, Koldijk M H, Cornelissen L A, Poon L L,    Peiris M, Koudstaal W, Wilson I A, Goudsmit J.

In one embodiment, in (b)(i) the J gene segments of the light chainlocus are J_(λ) gene segments and optionally the constant region of thelight chain locus is a lambda constant region; or in (b)(ii) the VL is aV_(λ) hand optionally the constant region of the light chain locus is alambda constant region. Alternatively, the constant region is C kappa.

In one embodiment, in (b)(i) the V gene segment repertoire of the lightchain locus comprises or consists of one or more VH gene segmentsselected from the group consisting of: a V_(H)III gene family member(optionally, a VHIIIa or VHIIIb family member), a V_(H)IV gene familymember, V_(H)III 9.1 (VH3-15), V_(H)III VH26 (VH3-23), V_(H)3-21,LSG6.1, LSG12.1, DP77 (V3-21), V_(H) H11, VH1GRR, ha3h2, V_(H)I-ha1c1,V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4,VH251, VH1-69 and a gene segment at least 80% identical thereto. Thesegene segments are useful because they expand the repertoire in vivo toVH gene segments that are found in natural human immune responses toantigens, such as antigens of infectious disease pathogens. This isuseful, for example, when the vertebrate is immunised with an antigen ofan infectious disease pathogen, for generation and isolation of anantibody for treating and/or preventing a disease or condition mediatedby said pathogen. In one example, in (b)(i) the V gene segmentrepertoire of the light chain locus comprises or consists of only VHgene segment selected from the group consisting of: a V_(H)III genefamily member (optionally, a VHIIIa or VHIIIb family member), a V_(H)IVgene family member, V_(H)III 9.1 (VH3-15), V_(H)III VH26 (VH3-23),V_(H)3-21, LSG6.1, LSG12.1, DP77 (V3-21), V_(H) H11, VH1GRR, ha3h2,V_(H)I-ha1c1, V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110,VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identicalthereto. This is useful to bias the immune response of the vertebrate(and thus resultant lead antibodies) to a predetermined gene segment,eg, one known to be commonly used in natural human immune responses toantigens, such as antigens of infectious disease pathogens. For example,VH1-69 is commonly used to produce antibodies in humans againstInfluenza virus (see Table 1); it is possible, therefore, to confine thesingle VH segment to VH1-69 in embodiment (b)(i) of the invention.

In one embodiment, in (b)(iii) the light chain locus V gene segmentrepertoire consists of only one (optionally only two, three or four) VLgene segment type (optionally and one or mutants thereof), wherein theVL gene segment is selected from said group of VL gene segments. This isuseful to bias the immune response of the vertebrate (and thus resultantlead antibodies) to a predetermined gene segment, eg, one known to becommonly used in natural human immune responses to antigens, such asantigens of infectious disease pathogens.

In one embodiment, in (a) said constant region is a heavy chainendogenous non-human vertebrate (optionally host mouse or rat) constantregion and/or in (b) said constant region is a light chain endogenousnon-human vertebrate (optionally host mouse or rat) constant region.

In one embodiment in any configuration of the invention, the genome hasbeen modified to prevent or reduce the expression of fully-endogenousantibody. Examples of suitable techniques for doing this can be found inPCT/GB2010/051122, U.S. Pat. Nos. 7,501,552, 6,673,986, 6,130,364,WO2009/076464, EP1399559 and U.S. Pat. No. 6,586,251, the disclosures ofwhich are incorporated herein by reference. In one embodiment, thenon-human vertebrate VDJ region of the endogenous heavy chainimmunoglobulin locus, and optionally VJ region of the endogenous lightchain immunoglobulin loci (lambda and/or kappa loci), have beeninactivated. For example, all or part of the non-human vertebrate VDJregion is inactivated by inversion in the endogenous heavy chainimmunoglobulin locus of the mammal, optionally with the inverted regionbeing moved upstream or downstream of the endogenous Ig locus (see, eg,WO2011004192, the disclosure of which is incorporated herein byreference). For example, all or part of the non-human vertebrate VJregion is inactivated by inversion in the endogenous kappa chainimmunoglobulin locus of the mammal, optionally with the inverted regionbeing moved upstream or downstream of the endogenous Ig locus. Forexample, all or part of the non-human vertebrate VJ region isinactivated by inversion in the endogenous lambda chain immunoglobulinlocus of the mammal, optionally with the inverted region being movedupstream or downstream of the endogenous Ig locus. In one embodiment theendogenous heavy chain locus is inactivated in this way as is one orboth of the endogenous kappa and lambda loci.

Additionally or alternatively, the vertebrate has been generated in agenetic background which prevents the production of mature host B and Tlymphocytes, optionally a RAG-1-deficient and/or RAG-2 deficientbackground. See U.S. Pat. No. 5,859,301 for techniques of generatingRAG-1 deficient animals.

Thus, in one embodiment of any configuration or aspect of the inventionherein, endogenous heavy and light chain expression has beeninactivated.

In a Second Configuration of the Invention, there is Provided

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

-   (a) An immunoglobulin heavy chain locus comprising one or more human    V gene segments (eg, a plurality of VH), one or more human D gene    segments and one or more human J gene segments upstream of a    constant region; and-   (b) (i) An unrearranged immunoglobulin light chain locus comprising    one or more human VH gene segments and one or more human J gene    segments upstream of a constant region, wherein each human VH gene    segment is a human gene segment identical to (or mutant of, eg,    having up to 15 or 10 nucleotide changes from the human gene    segment) a human VH gene segment (eg, a germline VH gene segment;    eg, a gene segment selected from List A1, A2, A1.1, A1.2, A2.1,    A2.2, A2.3 or A2.4 below) used to produce a rearranged VDJ encoding    a heavy chain variable region of a human antibody from an    antibody-expressing cell wherein said antibody binds to an antigen    of an infectious disease pathogen (optionally the variable regions    of said antibody being identical to an antibody from a human    individual suffering, susceptible to, or recovered from, a disease    or condition caused or mediated by an organism harbouring or    secreting said antigen; or from a human individual harbouring said    organism); or    -   (ii) An immunoglobulin light chain locus comprising a rearranged        VJ region or VDJ region upstream of a constant region, wherein        the nucleotide sequence of the recombined region is identical to        a nucleotide sequence produced by the recombination of a human J        gene segment and optionally a human D gene segment with a human        VH gene segment that is identical to (or mutant of; eg, having        up to 15 or 10 nucleotide changes from the human gene segment))        the human VH gene segment (eg, germline VH gene segment; eg, a        gene segment selected from List A1, A2, A1.1, A1.2, A2.1, A2.2,        A2.3 or A2.4 below) used to produce a rearranged VDJ encoding a        heavy chain variable region of a human antibody from an        antibody-expressing cell wherein said antibody binds to an        antigen of an infectious disease pathogen (optionally the        variable regions of said antibody being identical to an antibody        from a human individual suffering, susceptible to, or recovered        from, a disease or condition caused or mediated by an organism        harbouring or secreting said antigen; or from a human individual        harbouring said organism);-   (c) Wherein the gene segments in the heavy chain locus are operably    linked to the constant region thereof, and the gene segments or VJ    or VDJ in the light chain locus are operably linked to the constant    region thereof, so that upon immunisation the mouse is capable of    producing an antibody comprising heavy chains produced by    recombination of the heavy chain locus and light chains derived from    the light chain locus;-   (d) Optionally when (b)(i) applies, each said VH gene segment in the    light chain locus is selected from the group consisting of: a VHIII    gene family member (optionally, a VHIIIa or VHIIIb family member), a    VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),    VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2,    VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110, VH4.11,    71-4, VH251, VH1-69 and a gene segment at least 80% identical    thereto; optionally each VH gene segment is selected from List A1,    A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below.-   (e) Optionally when (b)(ii) applies, the nucleotide sequence of the    recombined region is identical to a nucleotide sequence produced by    the recombination of a human J gene segment and optionally a human D    gene segment with a human VH gene segment selected from the group    consisting of: a VHIII gene family member (optionally, a VHIIIa or    VHIIIb family member), a VHIV gene family member, VHIII 9.1    (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77    (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,    ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene    segment at least 80% identical thereto; optionally each VH gene    segment is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3    or A2.4 below.

In one embodiment, the antigen is an antigen expressed by a bacterial orviral infectious disease pathogen, eg, any of the pathogens listed inTable 1. For example, the antigen is an antigen selected from theantigens listed in Table 1.

In one embodiment of any aspect, configuration or embodiment of theinvention herein, the “human individual harbouring said organism” is apatient that has natural resistance to the pathogen and producesantibodies that bind to the pathogen or an antigen expressed thereby.

In one embodiment of the second configuration, the i gene segments ofthe light chain locus are J_(λ) gene segments and optionally theconstant region of the light chain locus is a lambda constant region.Alternatively, the constant region is C kappa.

In one embodiment of the second configuration, the V gene segmentrepertoire of the light chain locus comprises or consists of one or moreVH gene segments selected from the group consisting of: a V_(H)III genefamily member (optionally, a VHIIIa or VHIIIb family member), a V_(H)IVgene family member, V_(H)III 9.1 (VH3-15), V_(H)III VH26 (VH3-23),V_(H)3-21, LSG6.1, LSG12.1, DP77 (V3-21), V_(H) H11, VH1GRR, ha3h2,V_(H)I-ha1c1, V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1110,VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identicalthereto. These gene segments are useful because they expand therepertoire in vivo to VH gene segments that are found in natural humanimmune responses to antigens, such as antigens of infectious diseasepathogens. This is useful, for example, when the vertebrate is immunisedwith an antigen of an infectious disease pathogen, for generation andisolation of an antibody for treating and/or preventing a disease orcondition mediated by said pathogen. In one example, in (b)(i) the Vgene segment repertoire of the light chain locus comprises or consistsof only VH gene segment selected from the group consisting of: aV_(H)III gene family member (optionally, a VHIIIa or VHIIIb familymember), a V_(H)IV gene family member, V_(H)III 9.1 (VH3-15), V_(H)IIIVH26 (VH3-23), V_(H)3-21, LSG6.1, LSG12.1, DP77 (V3-21), V_(H) H11,VH1GRR, ha3h2, V_(H)I-ha1c1, V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051,71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least80% identical thereto. This is useful to bias the immune response of thevertebrate (and thus resultant lead antibodies) to a predetermined genesegment, eg, one known to be commonly used in natural human immuneresponses to antigens, such as antigens of infectious disease pathogens.For example, VH1-69 is commonly used to produce antibodies in humansagainst Influenza virus (see Table 1); it is possible, therefore, toconfine the single VH segment to VH1-69 in embodiment (b)(i) of theinvention.

In one embodiment of the second configuration, in (a) said constantregion is a heavy chain endogenous non-human vertebrate (optionally hostmouse or rat) constant region.

In one embodiment of the second configuration, in (b) said constantregion is a light chain endogenous non-human vertebrate (optionally hostmouse or rat) constant region.

In one embodiment of the second configuration, the genome of saidvertebrate or cell is homozygous for light chain locus (b)(i) or (ii);optionally wherein:

the V gene segment repertoire of the light chain loci consists of one ormore human VH gene segments selected from the group consisting of: aVHIII gene family member (optionally, a VHIIIa or VHIIIb family member),a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1,VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251,VH1-69 and a gene segment at least 80% identical thereto; or

the recombined VJ or VDJ repertoire of the light chain loci consists ofsequences identical to one or more nucleotide sequences produced by therecombination of a human VH gene segment selected from the groupconsisting of: a VHIII gene family member (optionally, a VHIIIa orVHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15),VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11,VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%identical thereto, with a human J gene segment and optionally a human Dgene segment. In one embodiment, all of the light chain locus V genesegments are from this group.

In one embodiment of the second configuration, endogenous heavy andlight chain expression has been inactivated, and wherein light chainloci according to the second configuration are the only functional lightchain loci in the genome of the vertebrate or cell.

In one embodiment of the second configuration, each immunoglobulin lightchain locus of said vertebrate or cell is according to (b)(i) andcomprises only a single human VH gene segment selected from the groupconsisting of: a VHIII gene family member (optionally, a VHIIIa orVHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15),VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11,VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2,Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80%identical thereto, optionally wherein the genome of the vertebrate orcell is homozygous for said light chain so that all light chain locicomprise the same, single human VH gene segment. In this embodiment (andgenerally in other embodiments, configurations and aspects of theinvention), confinement of heavy and/or light chain locus architectureis useful for biasing or controlling the antibody and gene repertoire,eg, to mirror human immune responses as mentioned above. Provision of asingle light or heavy chain variable (and optionally D and/or J) genesegment (or only this with closely related mutants thereof)—orconfinement in embodiments below to a single rearranged V(D)J region orsingle heavy or light chain—is advantageous for simplifying theexpression and production of therapeutic/prophylactic antibodies sincethis restricts the number of antibody species produced during downstreammanufacture. A common heavy or light chain is advantageous to enableco-expression of a plurality (eg, two, three or more) differentantibodies in the same expression medium, for example from the same hostcell. See, eg, EP1523496 (Merus B V) and WO2011097603 (RegeneronPharmaceuticals, Inc).

In one embodiment of the second configuration, each immunoglobulin lightchain locus of said vertebrate or cell is according to (b)(ii) andcomprises only a single rearranged VJ or VDJ region, optionally whereinthe genome of the vertebrate or cell is homozygous for said light chainso that all light chain loci comprise the same, single rearranged VJ orVDJ region.

In one embodiment of the second configuration, each immunoglobulin lightchain locus further comprises a VH gene segment or rearranged regionthat is a mutant (eg, having up to 15 or 10 nucleotide changes from theVH gene segment) respectively of said selected human VH gene segment orrearranged region, optionally wherein the genome of the vertebrate orcell is homozygous for said light chain mutant VH gene segment orrearranged region.

In one embodiment of the second configuration, each immunoglobulin lightchain locus comprises only two or three human VH gene segments selectedfrom said group, optionally wherein the genome of the vertebrate or cellis homozygous for said two or three light chain human VH gene segments.

In one embodiment of the second configuration, each immunoglobulin lightchain locus comprises only two or three of said rearranged VJ or VDJregions, optionally wherein the genome of the vertebrate or cell ishomozygous for said two or three light chain rearranged VJ or VDJregions.

The invention provides a monoclonal or polyclonal antibody compositionprepared by immunisation of at least one vertebrate (eg, mouse or rat)according to any configuration, aspect or embodiment of the invention,optionally wherein the antigen is an antigen of an infectious diseasepathogen (eg, a bacterial or viral pathogen antigen or an antigen listedin Table 1), optionally wherein the same antigen is used to immunise allthe vertebrates; optionally wherein the antibody or antibodies areIgG-type (eg, IgG1).

The invention provides a first method of isolating an antibody thatbinds a predetermined antigen (eg, a bacterial or viral pathogen antigenor an antigen listed in Table 1), the method comprising

-   (a) providing a vertebrate (optionally a mouse or rat) according to    according to any configuration, aspect or embodiment of the    invention;-   (b) immunising (eg, using a standard prime-boost method) said    vertebrate with said antigen (optionally wherein the antigen is an    antigen of an infectious disease pathogen);-   (c) removing B lymphocytes from the vertebrate and selecting one or    more B lymphocytes expressing antibodies that bind to the antigen;-   (d) optionally immortalising said selected B lymphocytes or progeny    thereof, optionally by producing hybridomas therefrom; and-   (e) isolating an antibody (eg, and IgG-type antibody) expressed by    the B lymphocytes.

In a first embodiment of the first method of the invention, the methodcomprises the step of isolating from said B lymphocytes nucleic acidencoding said antibody that binds said antigen; optionally exchangingthe heavy chain constant region nucleotide sequence of the antibody witha nucleotide sequence encoding a human or humanised heavy chain constantregion and optionally affinity maturing the variable region of saidantibody; and optionally inserting said nucleic acid into an expressionvector and optionally a host. The skilled person will be aware ofstandard molecular biology techniques to do this. For example, seeHarlow, E. & Lane, D. 1998, 5^(th) edition, Antibodies: A LaboratoryManual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and Pasqualiniand Arap, Proceedings of the National Academy of Sciences (2004)101:257-259 for standard immunisation. Joining of the variable regionsof an antibody to a human constant region can be effected by techniquesreadily available in the art, such as using conventional recombinant DNAand RNA technology as will be apparent to the skilled person. See e.g.Sambrook, J and Russell, D. (2001, 3′d edition) Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).

In one embodiment of the first method of the invention, the methodcomprises further making a mutant or derivative of the antibody.

A method of producing a polyclonal antibody mixture is provided, themethod comprising carrying out the first method of the invention byseparately immunising first and second vertebrates (optionally first andsecond mice or first and second rats) with antigen and combining theanti-antigen antibodies isolated from each vertebrate (or mutants orderivatives of said antibodies) to produce a polyclonal antibodymixture; optionally wherein the following apply separately or incombination ((i) and (ii); or (i) and (iii)):

(i) the vertebrates are immunised with the same antigen or differentantigens (optionally wherein the different antigens are expressed by thesame pathogenic organism (or by family members or different strains ofthe organism));(ii) prior to immunisation the light chain loci of the vertebratescontain the identical VH gene repertoire (optionally a single VH gene)and optionally the identical J repertoire; optionally the light chainloci of the mammals are identical prior to immunisation;(iii) prior to immunisation the light chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the light chain loci of the vertebratesare identical prior to immunisation.

A method of producing a polyclonal antibody mixture is provided, themethod comprising carrying out the first method of the invention byimmunising one or a plurality of vertebrates (optionally mice or rats)with first and second antigens and combining the anti-antigen antibodiesisolated from each vertebrate (or mutants or derivatives of saidantibodies) to produce a polyclonal antibody mixture; optionally whereinthe following apply separately or in combination ((i) and (ii); or (i)and (iii)):

(i) the antigens are expressed by the same pathogenic organism (or byfamily members or different strains of the organism));(ii) prior to immunisation the light chain loci of the vertebratescontain the identical VH gene repertoire (optionally a single VH gene)and optionally the identical J repertoire; optionally the light chainloci of the mammals are identical prior to immunisation;(iii) prior to immunisation the light chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the light chain loci of the vertebratesare identical prior to immunisation.

The Invention Provides a Second Method:

A method of producing host cells (eg, Chinese Hamster Ovary (CHO) orHEK293 cells) capable of expressing a polyclonal antibody mixture isprovided, the method comprising, in a method according to said firstembodiment of the first method of the invention:—

-   -   (a) immunising one or a plurality of vertebrates (optionally        mice or rats) with first and second antigens (optionally wherein        the different antigens are expressed by the same pathogenic        organism (or a family member thereof));    -   (b) isolating nucleic acid encoding first and second        anti-antigen antibodies from B lymphocytes from said        vertebrates;    -   (c) determining the nucleotide sequences of the heavy and light        chain variable regions (optionally the entire heavy and/or light        chain sequences) of the first antibody;    -   (d) determining the nucleotide sequence of the heavy variable        region and optionally the light chain variable region of the        second antibody;    -   (e) inserting the heavy chain variable region coding sequence of        each antibody into a heavy chain expression vector; optionally        wherein the constant region coding sequence of each heavy chain        is exchanged for a nucleotide sequence that encodes a human or        humanised constant region;    -   (f) inserting the light chain variable region coding sequence of        the first antibody into a light chain expression vector;        optionally wherein the constant region coding sequence of the        light chain of the first antibody is exchanged for a nucleotide        sequence that encodes a human or humanised constant region;    -   (g) optionally inserting the light chain variable region coding        sequence of the second antibody into a light chain expression        vector; optionally wherein the constant region coding sequence        of the light chain of the second antibody is exchanged for a        nucleotide sequence that encodes a human or humanised constant        region; and    -   (h) introducing each expression vector into a host cell and        co-expressing antibody chains in a mixture of said host cells to        produce antibodies, each antibody comprising one or both of said        heavy chain variable regions and a light chain; optionally        wherein the expression vectors are introduced together into the        same host cell (eg, a CHO or HEK293 cell) so that the cell is        capable of expressing antibody light chains and heavy chains,        such that the cell or a plurality of the host cells express        antibodies (eg, two, three, four or more different antibodies),        each comprising one or both of said heavy chain variable regions        and a light chain;    -   (i) optionally:        -   prior to immunisation the light chain loci of the            vertebrates contain the identical VH gene repertoire            (optionally a single VH gene segment) and optionally the            identical J repertoire (optionally a single J gene segment);            optionally the light chain loci of the vertebrates are            identical prior to immunisation; or prior to immunisation            the light chain loci of the vertebrates contain the            identical rearranged VJ or VDJ repertoire (optionally a            single VJ or VDJ); optionally the light chain loci of the            vertebrates are identical prior to immunisation.    -   (j) optionally:        -   producing a monoclonal or polyclonal antibody mixture, by            expressing a monoclonal antibody or polyclonal mixture of            said antibodies; optionally followed by isolating an            antibody comprising the heavy chain variable region of the            first and/or second antibodies.

The invention also provides a monoclonal or polyclonal antibody mixtureso produced or a derivative antibody or mixture thereof, eg, where oneor more constant region has been changed (eg, replaced with a differentconstant region such as a human constant region; or mutated to enhanceor ablate Fc effector function). (optionally the entire heavy and/orlight chain sequences)

In any of the methods of the invention, optionally each vertebrate usedfor immunisation is provided by

(a) isolating from a human blood or tissue (eg, B lymphocytes (PBLs),peripheral blood mononuclear cells (PBMCs), bone marrow, spleen, tonsilor lymph node) sample a B lymphocyte that expresses an antibody thatbinds a predetermined antigen (eg, an antigen expressed by an infectiousdisease pathogen; optionally wherein said serum or tissue was from ahuman individual suffering, susceptible to, or recovered from, a diseaseor condition caused or mediated by an organism harbouring or secretingsaid antigen; or from a human individual harbouring said organism); (b)determining which human germline VH gene segment was recombined in thehuman to produce the nucleotide sequence of said B lymphocyte thatencodes the heavy chain variable region of the antibody;(c) constructing a transgenic vertebrate wherein said human germline VHgene segment is provided in a light chain locus thereof according thefirst or second configuration of the invention; and(d) providing said transgenic vertebrate for immunisation in the firstmethod of the invention.

The term “Human blood” herein includes a human blood product minus oneor more non-B lymphocyte cellular populations, provided that the productretains antibody-producing cells, eg, PBLs.

In an embodiment of the first method of the invention, each vertebrateused for immunisation is provided by

(a) isolating from a human blood or tissue (eg, B lymphocytes, PBMCs,bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte thatexpresses an antibody that binds a predetermined antigen (eg, an antigenexpressed by an infectious disease pathogen; optionally wherein saidserum or tissue was from a human individual suffering, susceptible to,or recovered from, a disease or condition caused or mediated by anorganism harbouring or secreting said antigen; or from a humanindividual harbouring said organism);(b) determining a nucleotide sequence of said B lymphocyte that encodesa rearranged VDJ or VJ region of the antibody;(c) constructing a transgenic vertebrate wherein said rearranged VDJ orVJ region is provided in a light chain locus thereof according to thefirst or second configuration of the invention; and(d) providing said transgenic vertebrate for immunisation in the firstmethod of the invention.

Common Light Chain Antibodies & Bispecifics (eg, to Two PathogenAntigens for Infectious Diseases)

The invention provides an isolated antibody (eg, IgG-type, such asIgG1-type, antibody) obtainable or obtained by the second method of theinvention (including step (j), or a mutant or derivative antibodythereof wherein (i) the isolated antibody comprises two copies of theheavy chain variable region of said first antibody paired with twocopies of the light chain variable region of said first antibody; or(ii) the isolated antibody comprises two copies of the heavy chainvariable region of said second antibody paired with two copies of thelight chain variable region of said first antibody; or (iii) theisolated antibody is a bispecific antibody comprising one copy of theheavy chain variable region of said first antibody paired with a copy ofthe light chain variable region of the first antibody, and one copy ofthe heavy chain variable region of said the antibody paired with a copyof the light chain variable region of the first antibody, optionallywherein the bispecific antibody binds to said first and second antigensrecited in claim 24; optionally for use in medicine, optionally for thetreatment and/or prevention of an infectious disease.

In an aspect of the invention, there is provided a monoclonal orpolyclonal antibody mixture (eg, IgG-type antibody or antibodies),wherein the monoclonal antibody or mixture is according to anyconfiguration, aspect, embodiment or example of the invention, or amutant or derivative antibody thereof optionally for use in medicine,optionally for the treatment and/or prevention of an infectious disease,wherein optionally wherein each antibody binds an antigen of aninfectious disease pathogen, preferably the same antigen.

In an aspect of the invention, there is provided the use of an isolated,monoclonal or polyclonal antibody according to any configuration,aspect, embodiment or example of the invention, or a mutant orderivative antibody thereof in the manufacture of a medicament for thetreatment and/or prevention of an infectious disease, optionally whereinthe infectious disease is a disease caused by a bacterial or viralpathogen.

An example of a mutant antibody is one that bears up to 15 or 10 aminoacid mutations in its variable regions relative to an isolated antibody(eg, IgG-type, such as IgG1-type, antibody) obtainable or obtained bythe second method of the invention (including step (j). An example of aderivative is one that has been modified to replace a constant regionwith a different constant region such as a human constant region; ormutated to enhance or ablate Fc effector function.

Examples of infectious diseases are diseases caused or mediated by abacterial or viral pathogen, eg, a pathogen listed in Table 1. Examplesof antigens are those listed in Table 1.

For example, the infectious disease is selected from the groupconsisting of a disease caused by a pathogen selected from the groupconsisting of Haemophilus influenza, E. coli, Neisseria meningitidis, aherpes family virus, cytomegalovirus (CMV), HIV and influenza virus.

The invention further provides a nucleotide sequence encoding anantibody according to any configuration, aspect, embodiment or exampleof the invention, optionally wherein the nucleotide sequence is part ofa vector.

The invention further provides a pharmaceutical composition comprisingthe antibody or antibodies of any configuration, aspect, embodiment orexample of the invention and a diluent, excipient or carrier.

In a Third Configuration of the Invention, there is Provided

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

-   (a) An immunoglobulin heavy chain locus comprising either:—    -   (i) one or more human VL gene segments, one or more human D gene        segments and one or more human J gene segments upstream of a        constant region (optionally a rearranged V_(L)DJ_(H)C_(H) or        V_(λ)DJ_(H)C_(H)); or    -   (ii) one or more human VH gene segments selected from the group        consisting of: a VHIII gene family member (optionally, a VHIIIa        or VHIIIb family member), a VHIV gene family member, VHIII 9.1        (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77        (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIII-VH2-1, VH4.18,        ha4h3, Hv1051, 71-2, Hv1110, VH4.11, 71-4, VH251, VH1-69 and a        gene segment at least 80% identical; one or more human D gene        segments and one or more human J_(H) gene segments upstream of a        constant region; optionally each VH gene segment (and optionally        each D) is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2,        A2.3 or A2.4 below; and-   (b) An immunoglobulin light chain locus comprising one or more human    V gene segments (eg, a plurality of VL) and one or more human J gene    segments upstream of a constant region, optionally wherein the light    chain locus is according to (b)(i) or (b)(ii) of the first    configuration of the invention;

Wherein the gene segments in the heavy chain locus are operably linkedto the constant region thereof, and the gene segments in the light chainlocus are operably linked to the constant region thereof, so that uponimmunisation the mouse is capable of producing an antibody comprisingheavy chains produced by recombination of the heavy chain locus andlight chains produced by recombination of the light chain locus.

In one example, in (a)(i) all of the heavy chain locus V gene segmentsare human VL gene segments.

In one embodiment of the third configuration, the V gene segmentrepertoire of the light chain locus comprises or consists of one or moreVL gene segments selected from the group consisting of a V_(λ)II genefamily member, V_(λ)VII 4A, WI 2.1, V_(λ)VII 4A, a V_(λ)1 gene familymember, a V_(λ)3 gene family member, IGLV1S2, V_(λ)3-cML70, Ialh2,Ialvl, Ia3h3, Kv325, a VκI gene family member, κI-15A (KL012), V_(κ)IIfamily member, a V_(κ)III family member, a VκI gene family member,κI-15A (KL012), V_(κ)II A2 (optionally the Ata allele), V_(κ) A27(Humkv325) and a gene segment at least 80% identical thereto; optionallyeach VL gene segment is selected from List A1, A2, A1.1, A1.2, A2.1,A2.2, A2.3 or A2.4 below; and/or wherein or in (a)(ii) the heavy chainlocus V gene segment repertoire consists of only one (or two, three orfour) VH gene segment type (optionally and one or mutants thereof),wherein the VH gene segment is selected from said group of VH genesegments. This is useful to bias the immune response of the vertebrate(and thus resultant lead antibodies) to a predetermined gene segment,eg, one known to be commonly used in natural human immune responses toantigens, such as antigens of infectious disease pathogens.

In one embodiment of the third configuration, in (a) said constantregion is a heavy chain endogenous non-human vertebrate (optionally hostmouse or rat) constant region and/or in (b) said constant region is alight chain endogenous non-human vertebrate (optionally host mouse orrat) constant region.

In one embodiment of the third configuration, endogenous heavy and lightchain expression has been inactivated.

A Fourth Configuration of the Present Invention Provides

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cellwhose genome comprises:

-   (a) (i) An unrearranged immunoglobulin heavy chain locus comprising    one or more human VL gene segments, one or more human D gene    segments and one or more J_(H) gene segments upstream of a constant    region, wherein each human VL gene segment is a human gene segment    identical to (or mutant of, eg, having up to 15 or 10 nucleotide    changes from the human gene segment) a human VL gene segment (eg, a    germline VL gene segment; eg, a VL gene segment selected from List    A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below) used to produce    a rearranged VJ encoding a light chain variable region of a human    antibody from an antibody-expressing cell wherein said antibody    binds to an antigen of an infectious disease pathogen (optionally    the variable regions of said antibody being identical to an antibody    from a human individual suffering, susceptible to, or recovered    from, a disease or condition caused or mediated by an organism    harbouring or secreting said antigen; or from a human individual    harbouring said organism); or

(ii) An immunoglobulin heavy chain locus comprising a rearranged VJregion or VDJ region upstream of a constant region, wherein thenucleotide sequence of the recombined region is identical to anucleotide sequence produced by the recombination of a human J genesegment and optionally a human D gene segment with a human gene segmentidentical to (or mutant of, eg, having up to 15 or 10 nucleotide changesfrom the human gene segment) a human VL gene segment (eg, a germline VLgene segment; eg, a VL gene segment selected from List A1, A2, A1.1,A1.2, A2.1, A2.2, A2.3 or A2.4 below) used to produce a rearranged VJencoding a light chain variable region of a human antibody from anantibody-expressing cell wherein said antibody binds to an antigen of aninfectious disease pathogen (optionally the variable regions of saidantibody being identical to an antibody from a human individualsuffering, susceptible to, or recovered from, a disease or conditioncaused or mediated by an organism harbouring or secreting said antigen;or from a human individual harbouring said organism);

-   (b) An immunoglobulin light chain locus comprising one or more human    V gene segments (eg, a plurality of VL) and one or more human J gene    segments upstream of a constant region; and-   (c) Wherein the gene segments in the light chain locus are operably    linked to the constant region thereof, and the gene segments or VJ    or VDJ in the heavy chain locus are operably linked to the constant    region thereof, so that upon immunisation the mouse is capable of    producing an antibody comprising light chains produced by    recombination of the light chain locus and heavy chains derived from    the heavy chain locus;-   (d) Optionally when (a)(i) applies, each said VL gene segment in the    heavy chain locus is selected from the group consisting of a VL gene    segment selected from the group consisting of a VλII gene family    member, VλVII 4A, VλII 2.1, VλVII 4A, a VλI gene family member, a    Vλ3gene family member, IGLV1S2, Vλ3-cML70, Ialh2, Ialvl, Ia3h3,    Kv325, a VκI gene family member, κI-15A (KL012), VκII family member,    a VκIII family member, a VκI gene family member, κI-15A (KL012),    VκII A2 (optionally the A2a allele), VκA27 (Humkv325) and a gene    segment at least 80% identical thereto; optionally each VL gene    segment is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3    or A2.4 below;-   (e) Optionally when (a)(ii) applies, the nucleotide sequence of the    recombined region is identical to a nucleotide sequence produced by    the recombination of a human J gene segment and optionally a human D    gene segment with a human VL gene segment selected from the group    consisting of a VλII gene family member, VλVII 4A, VλII 2.1, VλVII    4A, a Vλ1 gene family member, a VX3gene family member, IGLV1S2,    VX3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a VκI gene family member,    κI-15A (KL012), VκII family member, a VκIII family member, a VκI    gene family member, κI-15A (KL012), VκII A2 (optionally the A2a    allele), Vκ A27 (Humkv325) and a gene segment at least 80% identical    thereto.

The group of VL gene segments is useful to bias the immune response ofthe vertebrate (and thus resultant lead antibodies) to a predeterminedgene segment, eg, one known to be commonly used in natural human immuneresponses to antigens, such as antigens of infectious disease pathogens.

In an embodiment of the fourth configuration, the VL gene segments ofthe heavy chain locus are V_(λ) gene segments.

In an embodiment of the fourth configuration, in (a) said constantregion is a heavy chain endogenous non-human vertebrate (optionally hostmouse or rat) constant region.

In an embodiment of the fourth configuration, in (b) said constantregion is a light chain endogenous non-human vertebrate (optionally hostmouse or rat) constant region.

In an embodiment of the fourth configuration, the genome of saidvertebrate or cell is homozygous for heavy chain locus (a)(i) or (ii);optionally wherein:

the V gene segment repertoire of the heavy chain loci consists of one ormore (or consists only of) human VL gene segments selected from thegroup consisting of a VL gene segment selected from the group consistingof a WI gene family member, V_(λ)VII 4A, V_(λ)II 2.1, V_(λ)VII 4A, aV_(λ)1 gene family member, a V_(λ)3 gene family member, IGLV1S2,V_(λ)3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a VκI gene family member,κI-15A (KL012), V_(κ)II family member, a V_(κ)III family member, a VκIgene family member, κI-15A (KL012), V_(κ)II A2 (optionally the A2aallele), V_(κ) A27 (Humkv325) and a gene segment at least 80% identicalthereto; or

the recombined VJ or VDJ repertoire of the heavy chain loci consists ofsequences identical to one or more nucleotide sequences produced by therecombination of a human VL gene segment selected from the groupconsisting of a VL gene segment selected from the group consisting of aVλII gene family member, VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene familymember, a Vλ3gene family member, IGLV1S2, Vλ3-cML70, Ialh2, Ialvl,Ia3h3, Kv325, a VκI gene family member, κI-15A (KL012), VκII familymember, a VκIII family member, a VκI gene family member, κI-15A (KL012),VκII A2 (optionally the A2a allele), Vκ A27 (Humkv325) and a genesegment at least 80% identical thereto with a human J gene segment andoptionally a human D gene segment.

In an embodiment of the fourth configuration, endogenous heavy and lightchain expression has been inactivated, and wherein heavy chain lociaccording to the fourth configuration are the only functional heavychain loci in the genome of the vertebrate or cell.

In an embodiment of the fourth configuration, each immunoglobulin heavychain locus of said vertebrate or cell is according to (a)(i) andcomprises only a single human VL gene segment selected from the groupconsisting of a VL gene segment selected from the group consisting of aVλII gene family member, VλVII 4A, VλII 2.1, VλVII 4A, a Vλ1 gene familymember, a Vλ3gene family member, IGLV1S2, Vλ3-cML70, Ialh2, Ialvl,Ia3h3, Kv325, a VκI gene family member, κI-15A (KL012), VκII familymember, a VκIII family member, a VκI gene family member, κI-15A (KL012),VκII A2 (optionally the Ata allele), Vκ A27 (Humkv325) and a genesegment at least 80% identical thereto, optionally wherein the genome ofthe vertebrate or cell is homozygous for said heavy chain so that allheavy chain loci comprise the same, single human VL gene segment.

In an embodiment of the fourth configuration, each immunoglobulin heavychain locus of said vertebrate or cell is according to (a)(ii) andcomprises only a single rearranged VJ or VDJ region, optionally whereinthe genome of the vertebrate or cell is homozygous for said heavy chainso that all heavy chain loci comprise the same, single rearranged VJ orVDJ region.

In an embodiment of the fourth configuration, each immunoglobulin heavychain locus further comprises a VL gene segment or rearranged regionthat is a mutant respectively of said selected human VL gene segment orrearranged region, optionally wherein the genome of the vertebrate orcell is homozygous for said light chain mutant VL gene segment orrearranged region.

In all configurations, aspects, examples and embodiments of theinvention, where a “mutant” is mentioned, this includes a mutantsequence that is identical to a reference sequence (eg, reference VH,VL, VJ or VDJ) but with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide oramino acid changes therefrom.

In an embodiment of the fourth configuration, each immunoglobulin heavychain locus comprises only two or three human VL gene segments selectedfrom said group, optionally wherein the genome of the vertebrate or cellis homozygous for said two or three heavy chain human VL gene segments.

In an embodiment of the fourth configuration, each immunoglobulin heavychain locus comprises only two or three of said rearranged VJ or VDJregions, optionally wherein the genome of the vertebrate or cell ishomozygous for said two or three heavy chain rearranged VJ or VDJregions.

The invention provides a monoclonal or polyclonal antibody compositionprepared by immunisation of at least one vertebrate (eg, mouse or rat)according to the third or fourth embodiment of the invention with anantigen, optionally wherein the antigen is an antigen of an infectiousdisease pathogen, optionally wherein the same antigen is used toimmunise all the vertebrates; optionally wherein the antibody orantibodies are IgG-type.

The invention provides a third method: A method of isolating an antibody(eg, IgG-type, such as IgG1) that binds a predetermined antigen, themethod comprising

(a) providing a vertebrate (optionally a mouse or rat) according to thethird or fourth embodiment of the invention;(b) immunising (eg, using standard prime-boost) said vertebrate withsaid antigen (optionally wherein the antigen is an antigen of aninfectious disease pathogen);(c) removing B lymphocytes from the vertebrate and selecting one or moreB lymphocytes expressing antibodies that bind to the antigen;(d) optionally immortalising said selected B lymphocytes or progenythereof, optionally by producing hybridomas therefrom; and(e) isolating an antibody (eg, and IgG-type antibody) expressed by the Blymphocytes;(f) Optionally, the third method comprises the step of isolating fromsaid B lymphocytes nucleic acid encoding said antibody that binds saidantigen; optionally exchanging the heavy chain constant regionnucleotide sequence of the antibody with a nucleotide sequence encodinga human or humanised heavy chain constant region and optionally affinitymaturing the variable region of said antibody; and optionally insertingsaid nucleic acid into an expression vector and optionally a host.

Optionally, the third method further comprises making a mutant orderivative of the antibody.

The invention provides a fourth method: A method of producing apolyclonal antibody mixture, the method comprising carrying out thethird method by separately immunising first and second vertebrates(optionally first and second mice or first and second rats) with antigen(eg, any antigen disclosed herein) and combining the anti-antigenantibodies isolated from each vertebrate (or mutants or derivatives ofsaid antibodies) to produce a polyclonal antibody mixture; optionallywherein the following apply separately or in combination ((i) and (ii);or (i) and (iii)):

(i) the vertebrates are immunised with the same antigen or differentantigens (optionally wherein the different antigens are expressed by thesame pathogenic organism (or different family members thereof ordifferent strains of the organism));(ii) prior to immunisation the heavy chain loci of the vertebratescontain the identical VL gene repertoire (optionally a single VL gene)and optionally the identical D and/or J repertoire; optionally the heavychain loci of the mammals are identical prior to immunisation;(iii) prior to immunisation the heavy chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the heavy chain loci of the vertebratesare identical prior to immunisation.

The invention provides a fifth method: A method of producing apolyclonal antibody mixture, the method comprising carrying out thethird method by immunising one or a plurality of vertebrates (optionallymice or rats) with first and second antigens and combining theanti-antigen antibodies isolated from each vertebrate (or mutants orderivatives of said antibodies) to produce a polyclonal antibodymixture; optionally wherein the following apply separately or incombination ((i) and (ii); or (i) and (iii)):

(i) the antigens are expressed by the same pathogenic organism (ordifferent family members thereof or different strains of the organism);(ii) prior to immunisation the heavy chain loci of the vertebratescontain the identical VL gene repertoire (optionally a single VL gene)and optionally the identical D and/or J repertoire; optionally the heavychain loci of the mammals are identical prior to immunisation;(iii) prior to immunisation the heavy chain loci of the vertebratescontain the identical rearranged VJ or VDJ repertoire (optionally asingle VJ or VDJ); optionally the heavy chain loci of the vertebratesare identical prior to immunisation.

The invention provides a sixth method: A method of producing host cellscapable of expressing a polyclonal antibody mixture, the methodcomprising, in the third method wherein step (f) is carried out:—

-   (a) immunising one or a plurality of vertebrates (optionally mice or    rats) with first and second antigens (optionally wherein the    different antigens are expressed by the same pathogenic organism (or    a family member thereof));-   (b) isolating nucleic acid encoding first and second anti-antigen    antibodies from B lymphocytes from said vertebrates;-   (c) determining the nucleotide sequences of the heavy and light    chain variable regions (optionally the entire heavy and/or light    chain sequences) of the first antibody;-   (d) determining the nucleotide sequence of the light variable region    and optionally the heavy chain variable region of the second    antibody;-   (e) inserting the light chain variable region coding sequence of    each antibody into a light chain expression vector; optionally    wherein the constant region coding sequence of each light chain is    exchanged for a nucleotide sequence that encodes a human or    humanised constant region;-   (f) inserting the heavy chain variable region coding sequence of the    first antibody into a heavy chain expression vector; optionally    wherein the constant region coding sequence of the heavy chain of    the first antibody is exchanged for a nucleotide sequence that    encodes a human or humanised constant region;-   (g) optionally inserting the heavy chain variable region coding    sequence of the second antibody into a heavy chain expression    vector; optionally wherein the constant region coding sequence of    the heavy chain of the second antibody is exchanged for a nucleotide    sequence that encodes a human or humanised constant region; and-   (h) introducing each expression vector into a host cell and    co-expressing antibody chains in a mixture of said host cells to    produce antibodies, each antibody comprising one or both of said    light chain variable regions and a heavy chain; optionally wherein    the expression vectors are introduced together into the same host    cell (eg, a CHO or HEK293 cell) so that the cell is capable of    expressing antibody light chains and heavy chains, such that the    cell or a plurality of the host cells express antibodies (eg, two,    three or four different antibodies), each comprising one or both of    said light chain variable regions and a heavy chain;-   (i) optionally:    -   prior to immunisation the heavy chain loci of the vertebrates        contain the identical VL gene repertoire (optionally a single VL        gene segment) and optionally the identical D and/or J repertoire        (optionally a single D and J gene segment); optionally the heavy        chain loci of the vertebrates are identical prior to        immunisation; or    -   prior to immunisation the heavy chain loci of the vertebrates        contain the identical rearranged VJ or VDJ repertoire        (optionally a single VJ or VDJ); optionally the heavy chain loci        of the vertebrates are identical prior to immunisation.

The invention also provides a monoclonal or polyclonal antibody mixtureso produced or a derivative antibody or mixture thereof, eg, where oneor more constant region has been changed (eg, replaced with a differentconstant region such as a human constant region; or mutated to enhanceor ablate Fc effector function).

The invention provides a seventh method: A method of producing amonoclonal antibody or polyclonal antibody mixture, the methodcomprising carrying out the sixth method and expressing a monoclonalantibody or polyclonal mixture of said antibodies; optionally followedby isolating an antibody comprising the light chain variable region ofthe first and/or second antibodies.

Optionally, each vertebrate used for immunisation is provided by

(a) isolating from a human blood or tissue (eg, B lymphocytes, PBMCs,bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte thatexpresses an antibody that binds a predetermined antigen (eg, an antigenexpressed by an infectious disease pathogen; optionally wherein saidserum or tissue was from a human individual suffering, susceptible to,or recovered from, a disease or condition caused or mediated by anorganism harbouring or secreting said antigen; or from a humanindividual harbouring said organism);(b) determining which human germline VL gene segment was recombined inthe human to produce the nucleotide sequence of said B lymphocyte thatencodes the light chain variable region of the antibody;(c) constructing a transgenic vertebrate wherein said human germline VLgene segment is provided in a heavy chain locus thereof according to thethird or fourth configuration of the invention; and(d) providing said transgenic vertebrate for immunisation in the fourth,fifth or sixth method of the invention.

In another embodiment, each vertebrate used for immunisation is providedby

(a) isolating from a human blood or tissue (eg, B lymphocytes, PBMCs,bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte thatexpresses an antibody that binds a predetermined antigen (eg, an antigenexpressed by an infectious disease pathogen; optionally wherein saidserum or tissue was from a human individual suffering, susceptible to,or recovered from, a disease or condition caused or mediated by anorganism harbouring or secreting said antigen; or from a humanindividual harbouring said organism);(b) determining a nucleotide sequence of said B lymphocyte that encodesa rearranged VDJ or VJ region of the antibody;(c) constructing a transgenic vertebrate wherein said rearranged VDJ orVJ region is provided in a heavy chain locus thereof according the thirdor fourth configuration of the invention; and(d) providing said transgenic vertebrate for immunisation in the methodof the fourth, fifth or sixth method of the invention.

Common Heavy Chain Antibodies & Bispecifics (eg, to Two PathogenAntigens for Infectious Diseases)

The invention provides an isolated antibody (eg, IgG-type antibody)obtainable or obtained by the seventh method, or a mutant or derivativeantibody thereof wherein (i) the isolated antibody comprises two copiesof the heavy chain variable region of said first antibody paired withtwo copies of the light chain variable region of said first antibody; or(ii) the isolated antibody comprises two copies of the heavy chainvariable region of said second antibody paired with two copies of thelight chain variable region of said first antibody; or (iii) theisolated antibody is a bispecific antibody comprising one copy of theheavy chain variable region of said first antibody paired with a copy ofthe light chain variable region of the first antibody, and one copy ofthe heavy chain variable region of said the antibody paired with a copyof the light chain variable region of the first antibody, optionallywherein the bispecific antibody binds to said first and second antigensdescribed above; optionally for use in medicine, optionally for thetreatment and/or prevention of an infectious disease.

The invention provides a monoclonal or polyclonal antibody mixture (eg,IgG-type antibody or antibodies), wherein the monoclonal antibody ormixture comprises or consists of antibodies produced by the fourth,fifth, sixth or seventh method, or a mutant or derivative antibodythereof optionally for use in medicine, optionally for the treatmentand/or prevention of an infectious disease, wherein optionally whereineach antibody binds an antigen of an infectious disease pathogen,preferably the same antigen.

The following embodiments relate to antibodies, host cells, nucleicacids and compositions and apply to such elements obtained or obtainableby any previous configuration or method of the invention:—

The invention provides an isolated chimaeric antibody for treatingand/or preventing an infectious disease or condition, the antibodycomprising a non-human vertebrate (optionally a mouse or rat) heavychain constant regions and human variable regions that bind an antigenof an infectious disease pathogen, wherein the antibody is obtainable orobtained in a method comprising immunisation of a vertebrate accordingto of any one of the first to seventh methods of the invention with saidantigen. The antigen is, for example, any antigen mentioned above. Thedisease or condition is, for example, any disease or condition mentionedabove.

The invention provides an isolated human antibody for treating and/orpreventing an infectious disease or condition, the antibody comprisinghuman heavy chain constant regions and human variable regions that bindan antigen of an infectious disease pathogen, wherein the antibody isobtainable or obtained in a method comprising affinity maturation ofantibody variable regions in vivo in a transgenic non-human vertebrate(eg, mouse or rat) when said variable regions are operably linked toheavy chain constant regions of said vertebrate (eg, mouse or rat heavychain constant regions) by (a) immunisation of a vertebrate of anyconfiguration of the invention with said antigen, (b) isolation ofnucleic acid encoding a chimaeric antibody as described above, (c)replacing the nucleotide sequences of the nucleic acid that encode thenon-human vertebrate heavy chain constant regions with nucleotidesequence encoding human heavy chain constant regions to produce nucleicacid encoding a human antibody; (d) expressing the human antibody invitro (optionally from CHO or HEK293 cells harbouring the human nucleicacid) and (e) isolating the human antibody (optionally with furtheraffinity maturation of the antibody and/or producing a derivativethereof). The invention provides a mixture of first and second suchhuman antibodies (an optionally also third and optionally fourthantibodies), each antibody being capable of binding to an antigen of aninfectious disease pathogen (optionally wherein the first antibody bindsa first antigen and the second antibody binds a second antigen, saidantigens being from the same pathogen; or wherein the antigens are thesame). Optionally, the light chain amino acid sequence of the firstantibody is identical to the light chain amino acid sequence of thesecond antibody, or has up to 15 amino acid changes therefrom. Theadvantages of such a common (or closely-related) chain are explainedabove, and include relative ease of manufacture.

The antigen is, for example, any antigen mentioned above. The disease orcondition is, for example, any disease or condition mentioned above. Thepathogen is, for example, any pathogen mentioned above.

The invention provides an antibody comprising human variable domainsthat bind a predetermined antigen (eg, an antigen expressed by abacterial or viral pathogen), wherein the variable domain sequences areencoded by rearranged VDJ and VJ regions, each of the VDJ and/or VJbeing a hybrid region produced by the in vivo rearrangement of humanheavy and light chain variable region gene segments (V and J andoptionally D segments); optionally wherein the antibody comprises humanconstant regions.

The invention provides a method of producing an isolated human antibodyfor treating and/or preventing an infectious disease or condition, theantibody comprising human heavy chain constant regions and humanvariable regions that bind an antigen of an infectious disease pathogen,wherein the method comprises affinity maturing antibody variable regionsin vivo in a transgenic non-human vertebrate (eg, mouse or rat) whensaid variable regions are operably linked to heavy chain constantregions of said vertebrate (eg, mouse or rat heavy chain constantregions) by (a) immunisation of a vertebrate of any configuration of theinvention with said antigen, (b) isolation of nucleic acid encoding achimaeric antibody as described above, (c) replacing the nucleotidesequences of the nucleic acid that encode the non-human vertebrate heavychain constant regions with nucleotide sequence encoding human heavychain constant regions to produce nucleic acid encoding a humanantibody; (d) expressing the human antibody in vitro (optionally fromCHO or HEK293 cells harbouring the human nucleic acid) and (e) isolatingthe human antibody (optionally with further affinity maturation of theantibody and/or producing a derivative thereof). The antigen is, forexample, any antigen mentioned above. The disease or condition is, forexample, any disease or condition mentioned above. The pathogen is, forexample, any pathogen mentioned above.

The invention provides the use of any isolated, monoclonal or polyclonalantibody or mixture of the invention as described above, in themanufacture of a medicament for the treatment and/or prevention of aninfectious disease, optionally wherein the infectious disease is adisease caused by a bacterial or viral pathogen. The disease orcondition is, for example, any disease or condition mentioned above. Thepathogen is, for example, any pathogen mentioned above. For example, theinfectious disease is selected from the group consisting of a diseasecaused by a pathogen selected from the group consisting of Haemophilusinfluenza, E. coli, Neisseria meningitidis, a herpes family virus,cytomegalovirus (CMV), HIV and influenza virus.

The invention provides first and second nucleotide sequences (eg, DNA,RNA, mRNA, cDNA) encoding the heavy and light chains of an antibodyaccording to any configuration, aspect, example or embodiment of theinvention or at least the variable regions thereof, optionally whereineach nucleotide sequence is part of a vector.

The invention provides a host cell comprising one or more expressionvectors encoding the heavy chains of the first and second antibodiesmentioned above, and the light chain of the first antibody mentionedabove (and optionally also the light chain of the second antibody).Again, reference is made to the discussion above about the advantages ofhaving a common antibody chain for the production of antibody mixtures.

The invention provides a pharmaceutical composition comprising theantibody or antibodies of any configuration, aspect, example orembodiment of the invention and a diluent, excipient or carrier;optionally wherein the composition is provided in a container connectedto an IV needle or syringe or in an IV bag. The skilled person will knowstandard diluents, excipients and carriers suitable for pharmaceuticalapplication.

Throughout this description, where it is mentioned “at least 80%identical”, there is contemplated in the alternative one of thefollowing identities: at least 85%, 90, 95, 96, 97, 98 or 99 identicaland the disclosure herein contemplates that one or more of theseidentities may be recited in a claim herein in place of “at least 80%identical”.

Tailoring V(D)J Incorporation into Immunoglobin Loci for the Generationof Antibodies Against Infectious Disease

In the various configurations, aspects, embodiments and examples above,the invention provides the skilled addressee with the possibility ofchoosing immunoglobulin gene segments in a way that tailors or biasesthe repertoire for application to generating antibodies to treat and/orprevent infectious diseases. The inventors have categorised thefollowing groups of gene segments for use in the invention according tothe desired application of resultant antibodies.

List A:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Pathogen

-   (a) a VL gene segment selected from the group consisting of a WI    gene family member, V_(λ)VII 4A, V_(λ)II 2.1, V_(λ)VII 4A, a V_(λ)1    gene family member, a V_(λ)3 gene family member, IGLV1S2,    V_(λ)3-cML70, Ialh2, Ialvl, Ia3h3, Kv325, a VκI gene family member,    κI-15A (KL012), V_(κ)II family member, a V_(κ)III family member, a    VκI gene family member, κI-15A (KL012), V_(κ)II A2 (optionally the    A2a allele), V_(κ) A27 (Humkv325) and a gene segment at least 80%    identical thereto.-   (b) a V_(λ) gene segment selected from a WI gene family member,    V_(λ)VII 4A, WI 2.1, V_(λ)VII 4A, a V_(λ)1 gene family member, a    V_(λ)3 gene family member, IGLV1S2, V_(λ)3-cML70, Ialh2, Ialvl,    Ia3h3 and a gene segment at least 80% identical thereto.-   (c) a V_(κ) gene segment selected from Kv325, a VκI gene family    member, κI-15A (KL012), V_(κ)II family member, a V_(κ)III family    member, a VκI gene family member, κI-15A (KL012), V_(κ)II A2    (optionally the A2a allele), V_(κ) A27 (Humkv325) and a gene segment    at least 80% identical thereto.-   (d) a V_(H) gene segment a V_(H)III gene family member (optionally,    a VHIIIa or VHIIIb family member), a V_(H)IV gene family member,    V_(H)III 9.1 (VH3-15), V_(H)III VH26 (VH3-23), V_(H)3-21, LSG6.1,    LSG12.1, DP77 (V3-21), V_(H) H11, VH1GRR, ha3h2, V_(H)I-ha1c1,    V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4,    VH251, VH1-69 and a gene segment at least 80% identical thereto.-   (e) a J_(λ) gene segment selected from J_(λ)2, J_(λ)3 and a gene    segment at least 80% identical thereto.-   (f) a D gene segment selected from Dk1, Dxp′1, Dn4r, D2r and a gene    segment at least 80% identical thereto.

List A1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Bacterial Pathogen

-   (a) a V_(λ) gene segment selected from a WI gene family member,    V_(λ)VII 4A, V_(λ)II 2.1, V_(λ)VII 4A and a gene segment at least    80% identical thereto.-   (b) a V_(κ) gene segment selected from a VκI gene family member,    κI-15A (KL012), V_(κ)II family member, a V_(κ)III family member, a    VκI gene family member, κI-15A (KL012), V_(κ)II A2 (optionally the    Ata allele), V_(κ) A27 (Humkv325) and a gene segment at least 80%    identical thereto.-   (c) a V_(H) gene segment a VH3 gene family member (optionally, a    VHIIIa or VHIIIb family member), V_(H)III 9.1 (VH3-15), V_(H)III    VH26 (VH3-23), V_(H)3-21, LSG6.1, LSG12.1, DP77 (V3-21), V_(H) H11    and a gene segment at least 80% identical thereto.-   (d) a J_(λ) gene segment selected from J_(λ)2, J_(λ)3 and a gene    segment at least 80% identical thereto.-   (e) a J_(H) gene segment selected from J_(H)2, J_(H)3, J_(H)4 and a    gene segment at least 80% identical thereto.

List A1.1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by H Influenza

-   (a) a V_(λ) gene segment selected from a WI gene family member,    V_(λ)VII 4A, V_(λ)II 2.1, V_(λ)VII 4A and a gene segment at least    80% identical thereto.-   (b) a V_(κ) gene segment selected from a V_(κ)II family member, a    V_(κ)III family member, a VκI gene family member, κI-15A (KL012),    V_(κ)II A2 (optionally the Ata allele), V_(κ) A27 (Humkv325) and a    gene segment at least 80% identical thereto.-   (c) a V_(H) gene segment a VH3 gene family member (optionally, a    VHIIIb family member), V_(H)III 9.1 (VH3-15), V_(H)III VH26    (VH3-23), V_(H)3-21, LSG6.1, LSG12.1, DP77 (V3-21) and a gene    segment at least 80% identical thereto.-   (d) a J_(λ) gene segment selected from J_(λ)2, J_(λ)3 and a gene    segment at least 80% identical thereto.

List A1.2:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by E Coli or Neisseria Meningitidis

-   (a) a V_(H) gene segment a VH3 gene family member (optionally a    VHIIIa or VHIIIb member), V_(H)III 9.1 (VH3-15), V_(H) H11, V_(H)III    VH26 (VH3-23) a gene segment at least 80% identical thereto, eg,    V_(H)III 9.1+J_(H)3; or V_(H) H11+J_(H)4; or V_(H)III VH26+J_(H)2.-   (b) a V_(κ) gene segment selected from a VκI gene family member,    κI-15A (KL012) and a gene segment at least 80% identical thereto.-   (c) a V_(λ) gene segment selected from a WI gene family member,    V_(λ)II 2.1 and a gene segment at least 80% identical thereto.-   (d) a J_(H) gene segment selected from J_(H)2, J_(H)3, J_(H)4 and a    gene segment at least 80% identical thereto.

A2:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by a Viral Pathogen

-   (a) a V_(H) gene segment selected from a V_(H)III gene family    member, a V_(H)IV gene family member, V_(H)III-VH26 (VH3-23),    VH1GRR, ha3h2, V_(H)I-ha1c1, V_(H)III-VH2-1, VH4.18, ha4h3, Hv1051,    71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at    least 80% identical thereto.-   (b) a V_(λ) gene segment selected from a V_(λ)1 gene family member,    a V_(λ)3 gene family member, IGLV1S2, V_(λ)3-cML70, Ialh2, Ialvl,    Ia3h3 and a gene segment at least 80% identical thereto.-   (c) a Vk gene segment selected from Kv325 and a gene segment at    least 80% identical thereto.-   (d) a J_(H) gene segment selected from J_(H)3, J_(H)S, J_(H)6 and a    gene segment at least 80% identical thereto.-   (e) a D gene segment selected from Dk1, Dxp′1, Dn4r, D2r and a gene    segment at least 80% identical thereto.-   (f) a J_(λ) gene segment selected from J_(λ)2, J_(λ)3 and a gene    segment at least 80% identical thereto.

A2.1:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by Herpes Virus Family (eg, VZV or HSV)

-   (a) a V_(H) gene segment selected from a V_(H)III gene family    member, a V_(H)IV gene family member, V_(H)III-VH26 (VH3-23),    VH1GRR, ha3h2, V_(H)I-ha1c1, V_(H)III-VH2-1, VH4.18, ha4h3, and a    gene segment at least 80% identical thereto.-   (b) a V_(λ) gene segment selected from a V_(λ)1 gene family member,    a V_(λ)3 gene family member, IGLV1S2, V_(λ)3-cML70, Ialh2, Ialvl,    Ia3h3 and a gene segment at least 80% identical thereto.-   (c) a J_(H) gene segment selected from J_(H)3, J_(H)S, J_(H)6 and a    gene segment at least 80% identical thereto.-   (d) a D gene segment selected from Dk1, Dxp′1, Dn4r, D2r and a gene    segment at least 80% identical thereto.-   (e) a J_(λ) gene segment selected from J_(λ)2, J_(λ)3 and a gene    segment at least 80% identical thereto. A2.2:    Immunoglobulin Gene Segments for Antibodies that Bind an Antigen    Expressed by CMV-   (a) a V_(H) gene segment selected from Hv1051 and a gene segment at    least 80% identical thereto.-   (b) a Vk gene segment selected from Kv325 and a gene segment at    least 80% identical thereto.

A2.3:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by HIV

-   (a) a V_(H) gene segment selected from 71-2, Hv1110, VH4.11, 71-4,    VH251, VH1-69 and a gene segment at least 80% identical thereto.

A2.4:

Immunoglobulin Gene Segments for Antibodies that Bind an AntigenExpressed by Influenza Virus

-   (a) a V_(H) gene segment selected from VH1-69 and a gene segment at    least 80% identical thereto.

Thus,

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease, one or more V, D and/or or all Jgene segments used in any configuration, aspect, method, example orembodiment of the invention can be selected from List A1. Thus, forexample in (a) of the first configuration of the invention, the recitedheavy chain V gene segment is selected from the VH gene segments in ListA, optionally with a D in that list.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by a bacterialpathogen, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by a viralpathogen, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by H. influenza,one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A1.1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by E Coli orNeisseria meningitidis, one or more or all V, D and/or J gene segmentsused in any configuration, aspect, method, example or embodiment of theinvention can be selected from List A1.2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by Herpes VirusFamily (eg, VZV or HSV), one or more or all V, D and/or J gene segmentsused in any configuration, aspect, method, example or embodiment of theinvention can be selected from List A2.1.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by CMV, one ormore or all V, D and/or J gene segments used in any configuration,aspect, method, example or embodiment of the invention can be selectedfrom List A2.2.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by HIV, one ormore or all V, D and/or J gene segments used in any configuration,aspect, method, example or embodiment of the invention can be selectedfrom List A2.3.

Where one wishes to generate an antibody or antibody mixture to treatand/or prevent an infectious disease caused or mediated by InfluenzaVirus, one or more or all V, D and/or J gene segments used in anyconfiguration, aspect, method, example or embodiment of the inventioncan be selected from List A2.4.

Optionally each VH segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each VL segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4

Optionally each D segment in the locus of the invention is selected fromList A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each J_(L) segment in the locus of the invention is selectedfrom List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Long HCDR3 Binding Sites & Tailoring Gene Segments to Pathogens & OtherAntigens

This aspect of the invention relates to the development of vertebrates,cells, methods and antibodies with relatively long HCDR3 binding sites.There is also provided embodiments in which genomes and antibodies aretailored in terms of their gene segments usage to address infectiousdisease pathogen antigens or other antigens which are advantageouslyaddressed with a longer HCDR3 length for binding or neutralisation.Antibodies may be raised in the vertebrates by immunisation with anon-pathogen target antigen, eg, an antigen bearing an epitope in acleft requiring a long CDR for contact, or an antigen from a pathogenthat causes or is implicated in harmful human disease or conditions.Examples are bacterial or viral pathogens and the target antigen may bea bacterial cell-surface antigen or a viral surface-exposed antigen (eg,coat protein). Additionally or alternatively, the antigen may be anantigen that is released (eg, secreted) from a pathogenic bacterium orvirus. The invention is not limited to addressing pathogen antigens, butis also useful for addressing other antigens where a long CDR3 would beuseful for binding (eg, an enzyme active site or receptor cleft).

Antibodies with long HCDR3 (at least 20 amino acids according to IMGTnomenclature) have been shown to effectively neutralise a variety ofpathogens including HIV, Influenza virus, malaria and Africatrypanosomes. Reference is also made to naturally-occurring Camelid (eg,llama or camel) heavy chain-only antibodies which bear long HCDR3s forreaching relatively inaccessible epitopes (see, eg, EP0937140). LongHCDR3s can form unique stable subdomains with extended loop structurethat towers above the antibody surface to confer fine specificity. Insome cases, the long HCDR3 itself is sufficient for epitope binding andneutralization (Liu, L et al; Journal of Virology. 2011. 85: 8467-8476,incorporated herein by reference). The unique structure of the longHCDR3 allows it to bind to cognate epitopes within inaccessiblestructure or extensive glycosylation on a pathogen surface. In humanperipheral blood, there is around 3.5% of naïve B antibodies or 1.9% ofmemory B IgG antibodies containing the HCDR3s with lengths of more than24 amino acids (Briney, B S et al, referenced given below) (FIG. 1 ofBriney, B S et al). The usage analysis indicates that these antibodieshave the preference to use human VH1-69, D2-2, D3-3, D2-15 and JH6segments (FIGS. 2-5 of Briney, B S et al). There are around 20% of allHCDR3 length antibodies using JH6. However, in those antibodies withmore than 24 amino acids of HCDR3, there are 70% using JH6 (FIG. 2 ofBriney, B S et al). Human VH5-51 is also commonly used for anti-HIVantibodies (see Gorny et al, PLoS One. 2011; 6(12):e27780. Epub 2011Dec. 2.

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VLlambda genes define a conserved antigenic structure, incorporated hereinby reference).

Supplementing these observations, the inventors have found (seeexamples) that other selected human heavy chain variable region genesegments (V, D, J) recombine in transgenic non-human vertebrates toproduce long HCDR3 (at least 20 amino acids).

Thus, as explained further in the examples, the inventors constructedtransgenic IgH loci in ES cells, wherein the loci purposely includedselected human heavy chain variable region gene segments (V, D, J) thatrecombine to produce long HCDR3 (at least 20 amino acids). From the EScells, the inventors generated transgenic non-human vertebrates (bothnaïve and immunised with a range of different target antigentypes—disease pathogen and human antigenic species), isolated antibodiesand heavy chain sequences based on the selected gene segments as well asB-cells expressing these and made hybridomas expressing antigen-specificantibodies that are based on the selected gene segments.

There is a need in the art for genetically modified non-human animalsthat prefer to make human antibodies that have long HCDR3s, as well asantibodies that can be selected from such animals wherein the antibodiescan address target epitopes more easily accessed by long HCDR3s. LongCDRH3 is also useful for penetrating highly glycan-covered epitope sites(eg, virus epitopes or any glycoprotein targets, eg, see Nature. 2011Dec. 14; 480(7377):324-5. doi: 10.1038/480324a; Vaccinology: “A sweetcleft in HIV's armour”, Sattentau Q J, incorporated herein byreference), and the target antigen can comprise such a target epitope.

The present invention provides vertebrates that can artificiallysimulate those naturally-occurring human long HCDR3 antibodies, and canprovide antibody, heavy chain and variable domain repertoires from whichcan be selected an antibody, heavy chain or variable domain having along HCDR3 (eg, having a HCDR3 length of 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more amino acids (according to IMGT). The inventionprovides for the combination of human VH, D and J gene repertoiresupstream of non-human vertebrate (eg, mouse or rat, eg, endogenous mouseor rat) constant region in heavy chain loci comprised by the vertebrategenomes. This enables the recombination, maturation and selection of thehuman gene segments in the context of endogenous or other non-vertebrateconstant regions which enhances the development of good sized antibody,heavy chain and variable domain repertoires from which to select longHCDR3-type binding sites. Thus, in an example of any configuration ofthe invention, the human gene segments are provided in a heavy chainlocus upstream of a non-human vertebrate (eg, endogenous) constantregion. Similarly any antibody of the invention comprises human variabledomains and non-human vertebrate (eg, endogenous) domains. The lattercan be replaced by human constant domains after selection and isolation.

For example, the following antibodies of the invention are contemplated(eg, produced in a vertebrate of this aspect of the invention by amethod disclosed herein) or a copy or derivative of an antibody soproduced:—

An isolated, synthetic or recombinant antibody comprising human heavychain variable domains having a HCDR3 length of 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more amino acids (according to IMGT), the heavychain variable domains being derived from the recombination of a humanVH gene segment selected from a VH group disclosed herein with a human Dgene segment and a human JH gene segment (optionally a JH6), wherein theantibody binds a target antigen; wherein the heavy chain variabledomains have non-human vertebrate AID-pattern somatic hypermutations,(eg, mouse or rat AID-pattern mutations) when compared to correspondinghuman germline V, D and J sequences and/or non-human (eg, mouse or rat)terminal deoxynucleotidyl transferase (TdT)-pattern junctional mutationswhen compared to corresponding human germline V, D and J sequences. Inan example, the antibody of the invention has a HCDR3 length of 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids (according to IMGT). Inan example, the antigen is an antigen of a pathogen that causes or isimplicated in a human infectious disease or condition, eg, a pathogenlisted in Table 1. In an example, the antibody specifically binds anactive site or cleft of an antigen (eg, an enzyme active site orreceptor cleft). This can be determined, eg, using standard X-raycrystallography of a complex of the antibody (or heavy chain or VHdomain) with the cognate antigen, as is known to the skilled person.

Mouse AID-pattern somatic hypermutations and/or mouse dTd-patternmutations can be provided, for example, wherein VH domain is produced ina mouse comprising mouse AID and/or mouse TdT (eg, endogenous AID orTdT). See also Annu. Rev. Biochem. 2007. 76:1-22; Javier M. Di Noia andMichael S. Neuberger, “Molecular Mechanisms of Antibody SomaticHypermutation” (in particular FIG. 1 and associated discussion on AIDhotspots in mouse); and Curr Opin Immunol. 1995 Apr.; 7(2):248-54,“Somatic hypermutation”, Neuberger M S and Milstein C (in particular,discussion on hotspots in mouse), the disclosures of which areincorporated herein by reference. Such mice can be made usingcorresponding mouse ES cell technology.

In an example, the antibody specifically binds to a HIV antigen. Severalnaturally-occurring human antibodies are known to be neutralising of HIVand have rather long HCDR3 lengths (20 amino acids or more according toIMGT; see Breden et al, PLoS One. 2011 Mar. 30; 6(3):e16857; “Comparisonof antibody repertoires produced by HIV-1 infection, other chronic andacute infections, and systemic autoimmune disease” (incorporated hereinby reference)—VH1-69 preferred for long HCDR3). See also PLoS One. 2012;7(5):e36750. Epub 2012 May 9; “Human peripheral blood antibodies withlong HCDR3s are established primarily at original recombination using alimited subset of germline genes”; Briney B S e al (incorporated hereinby reference). Thus, it is desirable to provide antibodies of theinvention that have similarly long HCDR3 lengths. The antibody of theinvention is, in one example, provided for treating and/or preventingHIV infection, eg, chronic HIV infection, in a human. The invention alsoprovides a method of treating and/or preventing HIV infection, eg,chronic HIV infection, in a human, the method comprising administering apharmaceutically acceptable dose of the antibody of the invention. Thedose can be split into one or more administration aliquots, eg,administered over a time course according to a medically-determinedregimen, as the skilled person will be able to determine.

In an example, the antibody specifically binds to Hemophilus influenzatype b polysaccharide. The antibody of the invention is, in one example,provided for treating and/or preventing Hemophilus influenza infection,eg, chronic Hemophilus influenza infection, in a human. The inventionalso provides a method of treating and/or preventing Hemophilusinfluenza infection, eg, chronic Hemophilus influenza infection, in ahuman, the method comprising administering a pharmaceutically acceptabledose of the antibody of the invention. The dose can be split into one ormore administration aliquots, eg, administered over a time courseaccording to a medically-determined regimen, as the skilled person willbe able to determine.

In an example, the antibody specifically binds to a rotavirus antigen(eg, protein 6 or 7). The antibody of the invention is, in one example,provided for treating and/or preventing rotavirus infection, eg, chronicrotavirus infection, in a human. The invention also provides a method oftreating and/or preventing rotavirus infection, eg, chronic rotavirusinfection, in a human, the method comprising administering apharmaceutically acceptable dose of the antibody of the invention. Thedose can be split into one or more administration aliquots, eg,administered over a time course according to a medically-determinedregimen, as the skilled person will be able to determine.

In an example, the antibody specifically binds to a cytomegalovirusantigen (eg, cytomegalovirus gB antigen). The antibody of the inventionis, in one example, provided for treating and/or preventingcytomegalovirus infection, eg, chronic cytomegalovirus infection, in ahuman. The invention also provides a method of treating and/orpreventing cytomegalovirus infection, eg, chronic cytomegalovirusinfection, in a human, the method comprising administering apharmaceutically acceptable dose of the antibody of the invention. Thedose can be split into one or more administration aliquots, eg,administered over a time course according to a medically-determinedregimen, as the skilled person will be able to determine.

The invention also provides a vertebrate or cell for expressing such anantibody; thus the invention provides a non-human vertebrate (eg, amouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or arat cell) whose genome comprises a human immunoglobulin VH gene segmentrepertoire that is biased to one, more or all human VH gene segmentsselected from a VH group disclosed herein.

The invention also provides a method of isolating an antibody that bindsa HIV antigen, Hemophilus influenza type b polysaccharide,cytomegalovirus antigen or rotavirus antigen, the method comprising

(a) providing the human VH biased vertebrate of the invention;(b) immunising said vertebrate with said HIV antigen, Hemophilusinfluenza type b polysaccharide, cytomegalovirus antigen or rotavirusantigen;(c) removing B lymphocytes from the vertebrate and selecting one or moreB lymphocytes expressing antibodies that bind to the antigen;(d) optionally immortalising said selected B lymphocytes or progenythereof, optionally by producing hybridomas therefrom; and(e) isolating an antibody (eg, and IgG-type antibody) expressed by the Blymphocytes, wherein the antibody has a HCDR3 length of 20 amino acidsor more.

Optionally, the method further comprises the step of isolating from saidB lymphocytes nucleic acid encoding said antibody that binds saidantigen; optionally exchanging the heavy chain constant regionnucleotide sequence of the antibody with a nucleotide sequence encodinga human or humanised heavy chain constant region and optionally affinitymaturing the variable region of said antibody; and optionally insertingsaid nucleic acid into an expression vector and optionally a host.

Optionally, the method further comprises making a copy, mutant orderivative (eg, humanised version) of the antibody produced by themethod.

This aspect of the invention also provides

A pharmaceutical composition comprising the anti-HIV antibody, fortreating and/or preventing HIV in a human (eg, an infant human).

A pharmaceutical composition comprising the anti-Hemophilus influenzatype b polysaccharide antibody, for treating and/or preventingHaemophilus influenza in a human (eg, an infant human).

A pharmaceutical composition comprising the anti-rotavirus antibody, fortreating and/or preventing rotavirus in a human (eg, an infant human).

A pharmaceutical composition comprising the anti-cytomegalovirusantibody, for treating and/or preventing cytomegalovirus in a human (eg,an infant human).

The invention also provides a method of generating such an antibody (eg,any one of embodiments (i) et seq above) by immunising a vertebrate ofthe invention with the target antigen and isolating the antibody fromthe vertebrate, optionally also making a copy or derivative of theantibody. In a further step, a B-cell capable of expressing the antibodyis isolated from the vertebrate. In a further step, a nucleic acidencoding the antibody (or a VH domain thereof) is isolated from thevertebrate (eg, a nucleic acid PCR cloned from a B-cell isolated fromthe vertebrate).

In an example, the antibody of the invention is a neutralising antibody.In an example, the antibody of the invention has a HCDR3 length of 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids (according toIMGT). In an example, the antibody of the invention has a HCDR3 lengthof 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids (accordingto IMGT). In an example, the antibody of the invention is isolated froma non-human vertebrate (eg, a mouse or a rat), for example a vertebrateof the invention; or the antibody is a copy or derivative (eg, humanisedversion) thereof. In an example, the antibody of the invention hasnon-human vertebrate constant regions (eg, mouse or rat constantregions); these may be replaced using standard recombinant DNAtechnology with human constant regions, so the invention also providesfor human versions of the antibodies recited above, wherein the humanantibody comprises human variable and constant regions, wherein thevariable regions bind the antigen. In an example, the antibody of thehas lambda-type human light chain variable domains. In another example,the antibody of the invention has kappa-type human light chain variabledomains.

Antibody competition can be determined, for example, by ELISA or surfaceplasmon resonance (SPR; eg, by competition Biacore™ or Proteon) as isstandard.

The invention also provides the following embodiments (recited below asnumbered clauses):—

D Bias

-   1. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell) whose genome    comprises a human immunoglobulin D gene segment repertoire that is    biased to the human D2 and/or D3 family or biased to one, more or    all human D gene segments selected from the group D1-26, D2-2, D3-9,    D3-10, D3-22, D4-17, D6-13 and D6-19.

For example, the repertoire consists of only human D gene segments fromthe D2 and/or D3 family.

Optionally the repertoire is biased to one or more of human D2-2, D2-15,D3-3, D3-9, D3-10 and D3-22, or the repertoire consists of one, more orall of these D gene segments. These produce long HCDR3 lengths (eg, seeTable 2 and references cited herein).

For example, the repertoire is biased to one or more of human ofD2-2*02, D3-9*01, D3-10*01 and D3-22*01, or the repertoire consists ofone, more or all of these D gene segments.

For example, the repertoire is biased to one or more of human D2-2*02,D3-9*01 and D3-10*01, or the repertoire consists of one, more or all ofthese D gene segments.

For example, the repertoire is biased to D3-9*01 and D3-10*01, orconsists of one, more or all of these D gene segments.

Optionally the repertoire consists of one, more or all of human D1-26,D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19. These produce longHCDR3 lengths (eg, see Table 2).

Optionally the repertoire is biased to one or more of human D1-26*01,D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01,or the repertoire consists of one, more or all of these D gene segments.

Optionally the repertoire is biased to one or more of human D2-2, D3-9,D3-10, D3-22, D4-17, D6-13 and D6-19, or the repertoire consists of one,more or all of these D gene segments. Optionally the repertoire isbiased to one or more of human D2-2*02, D3-9*01, D3-10*01, D3-22*01,D4-17*01, D6-13*01 and D6-19*01, or the repertoire consists of one, moreor all of these D gene segments. These produce long HCDR3 lengths innaïve repertoires (eg, see Table 2).

Optionally the repertoire is biased to one or more of human D1-26, D2-2,D3-10 and D6-19, or the repertoire consists of one, more or all of theseD gene segments. Optionally the repertoire is biased to one or more ofhuman D1-26*01, D2-2*02, D3-10*01 and D6-19*01, or the repertoireconsists of one, more or all of these D gene segments. These producelong HCDR3 lengths in immunised repertoires (eg, see Table 2).

Optionally the repertoire is biased to one or more of human D2-2, D3-9and D3-10, or the repertoire consists of one, more or all of these Dgene segments. Optionally the repertoire is biased to one or more ofhuman D2-2*02, D3-9*01 and D3-10*01, or the repertoire consists of one,more or all of these D gene segments. These produce long HCDR3 lengthsin antigen-specific repertoires (eg, see Table 2).

IMGT nomenclature is used for all gene segments.

Throughout this text, Genbank is a reference to Genbank release number185.0 or 191.0; the 1000 Genomes database is Phase 1, release v3, 16Mar. 2012; the Ensembl database is assembly GRCh37.p8 (10/04/2012); theIMGT database is available at www.imgt.org. The sequences of all VH genesegments explicitly mentioned herein are disclosed herein in theirentirety (for possible inclusion in clauses in conjunction with anyaspect of the invention as clauseed), such sequences being those in theIMGT and 1000 Genomes databases.

In one embodiment, the genome comprises an IgH locus comprising atargeted insertion of said human D gene segments. In an example, the IgHlocus comprises (in 5′ to 3′ order) one or more human VH gene segments,said D gene segment repertoire, one or more human JH gene segments and aconstant region (eg, wherein the constant region is a human constantregion or a non-human (eg, endogenous, eg, mouse or rat) constantregion).

In another embodiment, the genome comprises said human D gene segmentsrandomly inserted therein. This can be effected, eg, by incorporatinghuman DNA borne by YACS into the genome of ES cells (followed optionallyby generation of a non-human vertebrate therefrom, as is standard).

Optionally, the human D gene segment repertoire further comprises nomore than 5 additional human D gene segments, for example, therepertoire includes 1, 2, 3, 4 or 5 additional human D gene segments.

-   2. The vertebrate or cell of clause 1, wherein the D gene segment    repertoire consists of or substantially consists of one, two or    three human gene segments selected from the group D1-26, D2-2, D3-9,    D3-10, D3-22, D4-17, D6-13 and D6-19.-   3. The vertebrate or cell of clause 1 or 2, wherein the genome    comprises an unrearranged immunoglobulin heavy chain locus    comprising (in 5′ to 3′ order) human VH, D and JH gene segments and    said human D gene segments recited in clause 1 are spaced from the    VH gene segment(s) by no more than four other D gene segments (eg,    by no D gene segments).    -   This provides for bias wherein proximal D gene segments (those        more 3′, ie, closer to the constant region) are likely to be        more frequently used than those segments from distal (ie, 5′ or        further away from the constant region).-   4. The vertebrate or cell of any preceding clause, wherein the    genome comprises an unrearranged immunoglobulin heavy chain locus    comprising said human D gene segments and there are no other D gene    segments in the locus between said human D gene segments.    -   This is another way of biasing the repertoire of D gene        segments. Thus, the desired Ds are provided in tandem, aimed to        promote use in recombination.-   5. The vertebrate or cell of any preceding clause, wherein the    genome comprises three or more copies of a human D gene segment    selected from D1-26, D2-2, D2-15, D3-3, D3-9, D3-10, D3-22, D4-17,    D6-13 and D6-19.    -   For example, the genome comprises three or more copies of a        human D gene segment selected from D1-26, D2-2, D3-9, D3-10,        D3-22, D4-17, D6-13 and D6-19.    -   This is another way of biasing the repertoire of D gene        segments.-   6. The vertebrate or cell of clause 5, wherein the genome comprises    first and second human D gene segments selected from D1-26, D2-2,    D2-15, D3-3, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19 when the    first D gene segment is present as three or more copies and wherein    the second D gene segment is present as three or more copies.    -   For example, the first and second gene segments are selected        from D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19.    -   The various gene segment biasing techniques described herein can        be performed using conventional DNA manipulation in the        construction of transgenic vertebrates or cells of the        invention, which techniques (eg, recombineering and recombinant        DNA technology) will be known to the skilled person. For        example, BACs can be constructed using these techniques in which        the desired combination of human gene segments is provided, and        these BACs can be introduced into ES cells for incorporation of        the human gene segments into the genomes thereof (eg, by        targeted insertion into Ig loci). The ES cells can be used to        generate transgenic vertebrates as is standard and cells (eg,        B-cells) can be isolated from these wherein the genome is as per        the invention.

In one embodiment, the biased D gene segment(s) are selected from theIMGT database of variants or the 1000 Genomes database.

-   7. The vertebrate or cell of any preceding clause, wherein the D    gene segments are selected from D2-2*02, D3-9*01, D3-10*01 and    D3-22*01, or selected from D1-26*01, D2-2*02, D3-9*01, D3-10*01,    D3-22*01, D4-17*01, D6-13*01 and D6-19*01.

VH Bias

-   8. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell), optionally    according to any preceding clause, whose genome comprises a human    immunoglobulin VH gene segment repertoire that is biased to one,    more or all of gene segments selected from the group VH1-2, VH1-3,    VH1-8, VH1-18, VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13,    VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1.    -   These produce long HCDR3 lengths (see Table 2 and references        cited herein).    -   For example, the VH repertoire is biased to one, more or all of        VH1-2, VH1-3, VH1-8, VH1-18, VH2-5, VH3-7, VH3-9, VH3-11,        VH3-13, VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1. These        produce long HCDR3 lengths (see Table 2), or the repertoire        consists of one, more or all of these VH gene segments. For        example, the VH repertoire is biased to one, more or all of        VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,        VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,        VH6-1*01 and VH7-4-1*01, or the repertoire consists of one, more        or all of these VH gene segments.    -   For example, the VH repertoire is biased to one, more or all of        VH1-2*02, VH1-8*01, VH1-18*01, VH1-3*01, VH2-5*10, VH3-7*01,        VH3-9*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01        and VH7-4-1*01, or the repertoire consists of one, more or all        of these VH gene segments. These produce long HCDR3 lengths in        naïve repertoires (see Table 2).    -   For example, the VH repertoire is biased to one, more or all of        VH4-4*02, VH3-11*01 and VH3-7*01, or the repertoire consists of        one, more or all of these VH gene segments. These produce long        HCDR3 lengths in immunised repertoires (see Table 2).    -   For example, the VH repertoire is biased to one, more or all of        VH1-3*01, VH1-8*01, VH3-7*01, VH3-9*01, VH3-11*01 and VH4-4*02,        or the repertoire consists of one, more or all of these VH gene        segments. These produce long HCDR3 lengths in antigen-specific        repertoires (see Table 2).

Optionally, the human VH gene segment repertoire further comprises nomore than 5 additional human VH gene segments, for example, therepertoire includes 1, 2, 3, 4 or 5 additional human VH gene segments.

In one embodiment, the genome comprises an IgH locus comprising atargeted insertion of said human VH gene segments. In an example, theIgH locus comprises (in 5′ to 3′ order) said VH gene segment repertoire,one or more human D gene segments, one or more human JH gene segmentsand a constant region (eg, wherein the constant region is a humanconstant region or a non-human (eg, endogenous, eg, mouse or rat)constant region).

In another embodiment, the genome comprises said human VH gene segmentsrandomly inserted therein. This can be effected, eg, by incorporatinghuman DNA borne by YACS into the genome of ES cells (followed optionallyby generation of a non-human vertebrate therefrom, as is standard).

-   9. The vertebrate or cell of clause 8, wherein the VH gene segment    repertoire substantially consists of or substantially consists of    one, two or three human gene segments selected from VH1-2, VH1-3,    VH1-8, VH1-18, VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13,    VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4-1.    -   For example, the VH gene segment repertoire substantially        consists of or substantially consists of one, two or three human        gene segments selected from the group consisting of VH1-2*02,        VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01,        VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01        and VH7-4-1*01.-   10. The vertebrate or cell of clause 8 or 9, wherein the genome    comprises an unrearranged immunoglobulin heavy chain locus    comprising (in 5′ to 3′ order) human VH, D and JH gene segments and    said human VH gene segments are spaced from the D gene segment(s) by    no more than four other VH gene segments (eg, by no VH gene    segments).    -   This provides for bias wherein proximal VH gene segments (those        more 3′, ie, closer to the constant region) are likely to be        more frequently used than those segments from distal (ie, 5′ or        further away from the constant region).-   11. The vertebrate or cell of any one of clauses 8 to 10, wherein    the genome comprises an unrearranged immunoglobulin heavy chain    locus comprising said human VH gene segments and there are no other    VH gene segments in the locus between said human VH gene segments.    -   This is another way of biasing the repertoire of VH gene        segments.-   12. The vertebrate or cell of any one of clauses 8 to 11, wherein    the genome comprises three or more copies of a human VH gene segment    selected from the group consisting of VH1-2, VH1-3, VH1-8, VH1-18,    VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21,    VH3-23, VH4-4, VH6-1 and VH7-4-1.    -   For example, the genome comprises three or more copies of a        human VH gene segment selected from the group consisting of        VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,        VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,        VH6-1*01 and VH7-4-1*01.    -   This is another way of biasing the repertoire of VH gene        segments.-   13. The vertebrate or cell of clause 12, wherein the genome    comprises first and second human VH gene segments selected from the    group consisting of VH1-2, VH1-3, VH1-8, VH1-18, VH5-51, VH1-69,    VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4,    VH6-1 and VH7-4-1 when the first VH gene segment is present as three    or more copies and wherein the second VH gene segment is present as    three or more copies.    -   For example, the genome comprises first and second human VH gene        segments selected from the group consisting of VH1-2*02,        VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01,        VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01        and VH7-4-1*01 when the first VH gene segment is present as        three or more copies and wherein the second VH gene segment is        present as three or more copies.    -   In an embodiment, all or substantially all of VH gene segments        are present as three or more copies each.    -   The various gene segment biasing techniques described herein can        be performed using conventional DNA manipulation in the        construction of transgenic vertebrates or cells of the        invention, which techniques (eg, recombineering and recombinant        DNA technology) will be known to the skilled person. For        example, BACs can be constructed using these techniques in which        the desired combination of human gene segments is provided, and        these BACs can be introduced into ES cells for incorporation of        the human gene segments into the genomes thereof (eg, by        targeted insertion into Ig loci). The ES cells can be used to        generate transgenic vertebrates as is standard and cells (eg,        B-cells) can be isolated from these wherein the genome is as per        the invention.

In one embodiment, the biased D gene segment(s) are selected from theIMGT database of variants or the 1000 Genomes database.

-   14. The vertebrate or cell of any one of clauses 8 to 13, wherein    the VH gene segments are selected from the group consisting of    VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,    VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,    VH6-1*01 and VH7-4-1*01.    -   In an embodiment, the genome comprises a human immunoglobulin VH        gene segment repertoire that is biased to VH1-69.    -   In an embodiment, the human immunoglobulin VH gene segment        repertoire substantially consists of one or more human VH1-69        gene segments.    -   The gene segments are provided in one or more immunoglobulin        loci. For example, the gene segment repertoire (D and/or VH) is        provided in both IgH loci (ie, in a homozygous state).-   15. The vertebrate or cell of any one of clauses 8 to 16, comprising    an immunoglobulin heavy chain locus comprising two or more copies of    a VH gene segment selected from said group.    -   Thus, at least one of said copies is closer to the constant        region of the locus than the germline distance in a human from a        human constant region. The aim is to provide by bias by        providing more than one copy on the same locus. Also as at least        one of the copies is closer (more proximal to) the constant        region and J-C intron (which includes regulatory elements such        as the Emu enhancer region), this may favour use of the gene        segment, thus contributing to the desired bias.    -   Optionally, the genome is homozygous for the heavy chain locus.    -   Optionally the two or more copies of gene segments are identical        (eg, all VH1-69*01, using IMGT nomenclature). In another        example, copies are variants of each other, eg,        naturally-occurring human variants. Alternatively, synthetic        variants may be used with or without a naturally-occurring        variant.    -   In any embodiment of the invention, the vertebrate is naïve or        immunised with a target antigen.-   16. The vertebrate or cell of any clause, wherein the genome    comprises a human JH gene segment repertoire consisting of one or    more human JH6 gene segments.    -   This biases the JH repertoire for the production of long HCDR3,        since this is the longest naturally-occurring human JH gene        segment type and is commonly found in naturally-occurring human        antibodies having long HCDR3.    -   For example, the repertoire comprises two or more different JH6        variants. In an example, the repertoire comprises two or more        JH6*02 variants (IMGT nomenclature).-   17. The vertebrate or cell of any preceding clause, wherein the    genome comprises a human immunoglobulin JH gene segment repertoire    that is biased to JH6, optionally JH6*02.-   18. The vertebrate or cell of clause 17, wherein the JH gene segment    repertoire consists or substantially consists of three or more human    JH6 gene segments.-   19. The vertebrate or cell of any preceding clause, wherein the    sequence of each of said human gene segments is a human germline    gene segment sequence.-   20. The vertebrate or cell of any preceding clause, wherein one,    more or all of the selected gene segments are present in the genome    as two or more copies, the copies being variants of each other.    -   Thus, one, more or all of the human V, D and JH gene segments of        said genome is present in two or more variant versions, such as        naturally-occurring human variants, eg, variants found in the        1000 Genomes database and/or IMGT database. In another example,        one or more of the variants may be a synthetic variant.-   21. The vertebrate or cell of any preceding clause, wherein said    human gene segments are provided by homozygous immunoglobulin heavy    chain loci.    -   In an example, no other (non-human) active heavy chain VH, D or        JH gene segments are present in heavy chain loci of the genome.        Additionally, in an example no active non-human light chain VL        or JL gene segments are present in the genome.    -   This is useful for ensuring that endogenous (non-human) variable        region expression is inactivated. Thus, all heavy chains        produced by the vertebrate or cell will have human variable        regions, which is useful for producing drugs for administration        to humans.-   22. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell) whose genome    comprises a human immunoglobulin VH gene segment repertoire, one or    more human D gene segments and one or more human JH gene segments,    wherein the VH repertoire does not comprise one, more or all VH gene    segments selected from the group VH1-2, VH1-3, VH1-8, VH1-18,    VH5-51, VH1-69, VH2-5, VH3-7, VH3-9, VH3-11, VH3-13, VH3-20, VH3-21,    VH3-23, VH4-4, VH6-1 and VH7-4-1.-   23. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell) whose genome    comprises a human immunoglobulin D gene segment repertoire, one or    more human VH gene segments and one or more human JH gene segments,    wherein the D repertoire does not comprise one, more or all D gene    segments selected from the group D1-26, D2-2, D3-9, D3-10, D3-22,    D4-17, D6-13 and D6-19.    -   In instances it has been observed in that the art that certain        human gene usage may dominate the immune response to infectious        disease pathogen antigens or other antigens. While this may        yield many specific antibodies, typically these may not be        neutralising and thus the immune response is relatively        ineffective. This may happen, for example, where the antigen is        a decoy antigen expressed by the pathogen. The present        embodiments of the invention where specific gene segments are        omitted are useful for avoiding dominance of certain human gene        segments, such as those omitted from the genome. In this way,        the genome human gene segment repertoire is biased away from the        dominance and this enables better use and sampling of the        remaining human gene segment sequence space, thereby providing        the chance of producing antibodies that may not be normally        raised in a natural setting. Antigen specific antibodies can be        selected from vertebrates and cells with such genomes. In some        examples, this may yield neutralising antibodies.    -   It is advantageous to include a plurality of different human VH        gene segments, making up the human VH gene segment repertoire.        This provides for good diversities of rearranged human variable        regions from which to select leads. It is possible, for example,        to include an otherwise complete, functional repertoire of human        VH gene segments. To this end, the human VH gene segment        repertoire comprises, in one example, a plurality of human VH        gene segments, eg, at least 7, 10, 15, 20, 15, 30, 35, 40 or 45        different human VH gene segments. This can be achieved, for        example, using BACs harbouring stretches of unrearranged human        variable region DNA comprising VH gene segments—homologous        recombination and/or sRMCE being used to insert several        stretches of such DNA from serial BACs into an endogenous heavy        chain locus upstream of the constant region thereof in the        genome of a non-human vertebrate ES cell (eg, mouse or rat ES        cell), followed by development of one or more progeny        vertebrates from such cells (and optional breeding to        homozygosity of the heavy chain locus). In one embodiment, human        DNA is inserted that includes a first human VH (eg, VH1-69        and/or VH1-2) and flanking VH gene segments upstream and        downstream of these. In a second ES cell genomic manipulation,        the first VH is deleted from the genome, eg, using standard        homolgous recombination techniques as is known in the art. In        this way, one or more VH gene segments usually upstream and/or        downstream of the deleted gene segment(s) in a wild-type human        germline genome are retained so that they can be available to        contribute to the subsequent rearranged human V region        repertoire that is used for selection of leads. In another        example, the initial insertion of human DNA is made using        stretches of DNA that already omit the first VH (eg, by deleting        such stretches using recombineering of BACs in E. coli, as is        known in the art). Similar techniques can be used (with        appropriate BACs) for the omission of human D and/or J gene        segments.

Thus, in an embodiment, VH gene segments that normally are upstreamand/or downstream of the omitted human VH gene segments (or omitted D orJ as per other embodiments) in a wild-type germline human genome areincluded in the vertebrate or cell of the invention. For example, thehuman VH gene segment repertoire of the genome in the invention does notcomprise VH1-69, but does comprise one, two, three or four human VH genesegments selected from VH2-10, VH3-72, VH3-73 and VH3-74. These are genesegments that are immediately upstream of VH1-69 in a wild-type humangermline heavy chain locus (see IMGT). For example, additionally oralternatively the human VH gene segment repertoire of the genome in theinvention does not comprise VH1-69, but does comprise one, two, three,four or more (or all of) human VH gene segments selected from VH3-66,VH3-64, VH4-61, VH4-59, VH1-58, VH3-53, VH3-49, VH3-48, VH1-46 andVH1-45. These are gene segments that are immediately downstream ofVH1-69 in a wild-type human germline heavy chain locus (see IMGT).Additionally or alternatively, the human VH gene segment repertoire ofthe genome in the invention does not comprise VH1-69, but does compriseone, two, three, four or more (or all of) human VH gene segmentsselected from VH2-5, 7-41, 4-4, 1-3, 1-2 and 6-1. Additionally oralternatively, the human VH gene segment repertoire of the genome in theinvention does not comprise VH1-69, but does comprise one, two, three,four, 5, 6, 7, 8, 9, 10 or more (or all of) human VH gene segmentsselected from VH2-5, 7-41, 4-4, 1-3, 1-2, 6-1, 3-7, 1-8, 3-9, 3-11 and3-13. Additionally or alternatively, the human VH gene segmentrepertoire of the genome in the invention does not comprise VH1-69, butdoes comprise one, two, three, four, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17 or more (or all of) human VH gene segments selected fromVH2-5, 7-41, 4-4, 1-3, 1-2, 6-1, 3-7, 1-8, 3-9, 3-11, 3-13, 3-15, 1-18,3-20, 3-21, 3-23, 1-24 and 2-26. Additionally or alternatively, thehuman VH gene segment repertoire of the genome in the invention does notcomprise VH1-69, but does comprise VH6-1 (which is commonly used inhuman immune responses, VH6-1 being the most proximal to the constantregion in a wild-type human germline heavy chain locus) and/or VH3-23(which is commonly used in human immune responses). In embodiment (eg,for generating VH, heavy chains or antibodies for treating and/orpreventing an infectious disease, eg, HIV infection, in a human), VH1-2is omitted in the genome or locus. In this case one, two, three or allhuman VH gene segments immediately 5′ and 3′ of VH1-2 in a wild-typegermline human IgH locus (eg, see IMGT) are included in the genome, suchas comprised by the same IgH locus upstream of human D and JH genesegments and a constant region.

-   24. The vertebrate or cell of clause 22 or 23, wherein the genome    comprises a human JH gene segment repertoire that does not comprise    JH6.

JH Bias

-   25. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell) whose genome    comprises a human immunoglobulin JH gene segment repertoire that is    biased to human JH6.    -   In an example, the repertoire is biased to human JH6*02 (IMGT        nomenclature).    -   So, the inventors made a choice of human JH6*02 on the basis of    -   (i) Containing YYG and YYGXDX motifs that is conserved across        several vertebrate species;    -   (ii) Provision of one less TAC codon than other human JH6        variant (an AID hotspot that risks stop codons) and a choice        instead of a codon that preserves the YYG and YYGXDX motifs;    -   (iii) Avoidance of a GGCA AID hotspot in the region of the        HCDR3/FW4 junction; and    -   (iv) Common occurrence (and thus conservation and acceptability)        in humans of the JH6*02 variant.-   26. The vertebrate or cell of clause 25, wherein the genome    comprises an unrearranged immunoglobulin heavy chain locus    comprising a plurality of human JH6 gene segments; optionally    wherein the genome is homozygous for said locus.    -   In an example, the plurality comprises or consists of a        plurality of JH6*02 gene segments.-   27. The vertebrate or cell of clause 26, wherein the heavy chain    locus comprises (in 5′ to 3′ order) human VH, D and JH gene segments    and said JH6 gene segments are spaced from the D gene segment(s) by    no more than two other JH gene segments.-   28. The vertebrate or cell of clause 25, 26 or 27, wherein are no    other JH gene segments in the locus between said human JH6 gene    segments.-   29. A non-human vertebrate (eg, a mouse or a rat) or a non-human    vertebrate cell (eg, a mouse cell or a rat cell) whose genome    comprises a human immunoglobulin JH gene segment repertoire that    consists of one or more human JH6 gene segments.    -   In an example, all of the gene segments are JH6*02 gene        segments.-   30. The vertebrate or cell of any one of clauses 25 to 29, wherein    all of said gene segments are human germline gene segments.-   31. The vertebrate or cell of any one of clauses 25 to 30,    comprising different variant JH6 gene segments.    -   In an example, the variants are all naturally-occurring (eg,        appearing in the IMGT or 1000 Genome databases). In an other        example, one or more variant is synthetic.-   32. The vertebrate or cell of any one of clauses 25 to 31, wherein    said gene segments are provided by homozygous immunoglobulin heavy    chain loci.

In one embodiment, the biased JH gene segment(s) are selected from theIMGT database of variants or the 1000 Genomes database.

-   33. A monoclonal or polyclonal antibody composition or a population    of antibody-producing cells for producing such composition, wherein    the composition or population is prepared by immunising at least one    vertebrate according to any preceding clause with an antigen,    wherein the antibody or antibodies have human heavy chain variable    regions comprising non-human vertebrate AID-pattern somatic    hypermutations, (eg, mouse or rat AID-pattern mutations) when    compared to corresponding human germline V, D and J sequences and/or    non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase    (TdT)-pattern junctional mutations when compared to corresponding    human germline V, D and J sequences; wherein the composition    comprises at least one antigen-specific antibody having a HCDR3    length of at least 20 amino acids (according to IMGT).    -   As will be readily apparent to the skilled person, AID and TdT        mutations can be determined using bioinformatics analysis to        find the closest matching human germline gene segment(s) that        correspond to a given variable domain sequence, aligning the        sequences and determining the differences. AID has known        hotspots for mutation (eg, see Annu. Rev. Biochem. 2007.        76:1-22; Javier M. Di Noia and Michael S. Neuberger, “Molecular        Mechanisms of Antibody Somatic Hypermutation” (in particular        FIG. 1 and associated discussion on AID hotspots in mouse); and        Curr Opin Immunol. 1995 April; 7(2):248-54, “Somatic        hypermutation”, Neuberger M S and Milstein C (in particular,        discussion on hotspots in mouse), the disclosures of which are        incorporated herein by reference). By carrying out the standard        bioinformatics analysis, TdT mutations (eg, to provide        junctional mutations and diversity) can be determined, as will        be familiar to the skilled person.    -   Corresponding human germline V, D and J sequences can be        according to the IMGT database or 1000 Genomes database, for        example.    -   For example, the HCDR3 length is at least 20, 21, 22, 23, 24,        25, 26, 27, 28, 29 or 30 amino acids.    -   For example, the HCDR3 length is from 20 to 23 or 24 to 30, eg,        from 28 to 30 amino acids.    -   For example, the cells are B cells (eg, immortalised B cells) or        hybridomas.    -   Optionally the antibodies of any aspect of the invention        comprise human light chain variable regions. For example, the        human light chain variable regions have non-human vertebrate        AID-pattern somatic hypermutations, (eg, mouse or rat        AID-pattern mutations) when compared to corresponding human        germline V, D and J sequences and/or non-human (eg, mouse or        rat) terminal deoxynucleotidyl transferase (TdT)-pattern        junctional mutations when compared to corresponding human        germline V, D and J sequences.-   34. An isolated antibody that specifically binds an antigen, the    antibody comprising human heavy chain variable regions and non-human    constant regions, wherein the variable regions are derived from the    recombination in a non-human vertebrate of (i) a human VH gene    segment selected from the group recited in clause 8 with (ii) a    human D gene segment selected from the group recited in clause 1 and    with a human JH gene segment (optionally JH6); wherein the antibody    has a HCDR3 length of at least 20 amino acids (according to IMGT);    and non-human vertebrate AID-pattern somatic hypermutations, (eg,    mouse or rat AID-pattern mutations) when compared to corresponding    human germline V, D and J sequences and/or non-human (eg, mouse or    rat) terminal deoxynucleotidyl transferase (TdT)-pattern junctional    mutations when compared to corresponding human germline V, D and J    sequences.    -   In examples, the VH is selected from the group VH1-2*02,        VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01,        VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01        and VH7-4-1*01 and/or    -   the D is selected from the group    -   D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or    -   D2-2*02, D3-9*01 and D3-10*01, or    -   D3-9*01 and D3-10*01, or    -   D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or    -   D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01,        D6-13*01 and D6-19*01, or    -   D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D2-2*02,        D3-9*01, D3-10*01, D3-22*01,    -   D4-17*01, D6-13*01 and D6-19*01, or    -   D1-26, D2-2, D3-10 and D6-19, or    -   D2-2, D3-9 and D3-10.-   35. The antibody of clause 34, wherein the antibody is obtained or    obtainable from a vertebrate according to any one of clauses 1 to    32.    -   In an embodiment, the antibody is obtained from said vertebrate,        or is a copy of such an antibody.-   36. A method of isolating an antibody that binds a predetermined    antigen, the method comprising    -   (a) providing a vertebrate (optionally a mouse or rat) according        to any one of clauses 1 to 32;    -   (b) immunising said vertebrate with said antigen;    -   (c) removing B lymphocytes from the vertebrate and selecting one        or more B lymphocytes expressing antibodies that bind to the        antigen;    -   (d) optionally immortalising said selected B lymphocytes or        progeny thereof, optionally by producing hybridomas therefrom;        and    -   (e) isolating an antibody (eg, and IgG-type antibody) expressed        by the B lymphocytes.-   37. The method of clause 36, wherein in step (e) wherein the    antibody has a HCDR3 length of at least 20 amino acids (according to    IMGT).    -   The length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30        amino acids (according to IMGT), eg, from 20 to 23 amino acids        (a produced in the examples).-   38. The method of clause 36 or 37, comprising the step of isolating    from said B lymphocytes nucleic acid encoding said antibody that    binds said antigen; optionally exchanging the heavy chain constant    region nucleotide sequence of the antibody with a nucleotide    sequence encoding a human or humanised heavy chain constant region    and optionally affinity maturing the variable region of said    antibody; and optionally inserting said nucleic acid into an    expression vector and optionally a host.-   39. The method of clause 36, 37 or 38, further comprising making a    copy, mutant or derivative (eg, humanised version) of the antibody    produced by the method.    -   Humanisation can entail making the constant regions human.-   40. The antibody composition, cell population, antibody or method of    any one of clauses 33 to 39, wherein the antigen is an antigen of an    infectious disease pathogen; optionally wherein the pathogen is a    virus or bacterium.-   41. The antibody composition, cell population, antibody or method of    clause 40, wherein pathogen is selected from the group consisting of    Haemophilus influenza, E. coli, Neisseria meningitidis, a herpes    family virus, cytomegalovirus (CMV), HIV and influenza virus.-   42. The antibody composition, cell population, antibody or method of    any one of clauses 33 to 41, wherein the antigen is a HIV gp120    antigen or a HIV gp41 antigen.-   43. The antibody composition, cell population, antibody or method of    any one of clauses 33 to 40, wherein the antigen comprises an active    site or cleft, wherein the antibody having a HCDR3 length of at    least 20 amino acids specifically binds to the active site or cleft    of the antigen.-   44. A pharmaceutical composition comprising an antibody or antibody    composition according to any one of clauses 33 to 35 and 40 to 43,    or an antibody produced by the method of any one of clauses 36 to    38, for treating and/or preventing an infectious disease in a human    (eg, wherein the infectious disease is caused by a pathogen selected    from the group consisting of Haemophilus influenza, E. coli,    Neisseria meningitidis, a herpes family virus, cytomegalovirus    (CMV), HIV and influenza virus).-   45. A repertoire of antibody heavy chains (eg, provided by    antibodies) comprising one or more heavy chains whose variable    domain HCDR3 has a length of at least 20 amino acids (according to    IMGT) and derived from the recombination of a human VH, D and JH,    wherein    -   the VH is selected from the group    -   VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01,        VH3-9*01, VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02,        VH6-1*01 and VH7-4-1*01 and    -   the D is selected from the group    -   D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or    -   D2-2*02, D3-9*01 and D3-10*01, or    -   D3-9*01 and D3-10*01, or    -   D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or    -   D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01,        D6-13*01 and D6-19*01, or    -   D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D2-2*02,        D3-9*01, D3-10*01, D3-22*01,    -   D4-17*01, D6-13*01 and D6-19*01, or    -   D1-26, D2-2, D3-10 and D6-19, or    -   D2-2, D3-9 and D3-10;    -   and optionally the JH is JH6 (eg, JH6*02);    -   Wherein    -   (a) the heavy chain variable domain has been produced in vivo in        a non-human vertebrate (eg, a mouse or a rat); and/or    -   (b) the heavy chain variable domain comprises non-human        vertebrate AID-pattern somatic hypermutations, (eg, mouse or rat        AID-pattern mutations) when compared to corresponding human        germline V, D and J sequences and/or non-human (eg, mouse or        rat) terminal deoxynucleotidyl transferase (TdT)-pattern        junctional mutations when compared to corresponding human        germline V, D and J sequences.    -   In an example, the heavy chain (or all heavy chains in the        repertoire) comprise non-human vertebrate constant regions (eg,        mouse or rat constant regions). For example, the constant        regions are gamma-type constant regions (eg, gamma-1, gamma-2 or        gamma-4 type).    -   In an example, the repertoire is a naïve repertoire. This is        shown in the examples section herein.    -   In an example, the repertoire is an immunised repertoire. This        is shown in the examples section herein.    -   In an example, the repertoire is an antigen-specific repertoire        (eg, provided by a plurality of hybridomas). This is shown in        the examples section herein.    -   The repertoire can be provided by B cells (eg, immortalised B        cells).    -   The repertoire can be provided by hybridomas.    -   In an example, the vectors are harboured by host cells (eg, CHO        or HEK293 cells or yeast cells).    -   The HCDR3 length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29        or 30 amino acids (according to IMGT), eg, from 20 to 23 amino        acids (a produced in the examples).    -   In an example, in (a) the vertebrate is a vertebrate according        to the invention.-   46. A nucleic acid collection encoding the heavy chain repertoire of    clause 45.    -   In an example, the nucleic acids are provided in respective        vectors (eg, expression vectors, eg, E coli or CHO or HEK293        vectors).-   47. A method of obtaining an antigen-specific heavy chain (eg,    provided by an antibody), the method comprising exposing the    repertoire of clause 45 to a predetermined antigen and selecting one    or more heavy chains that specifically bind to the antigen, wherein    one or more heavy chains is isolated that has a HCDR3 length of at    least 20 amino acids.    -   Optionally, when the heavy chain has a non-human constant        region, this is swapped for a human constant region, as is        conventional in the art. Thus, the invention provides a human        antibody heavy chain so produced (eg, provided in combination        with a human light chain to produce a human antibody which is        useful for human therapeutic and/or prophylactic use, eg, to        treat and/or prevent an infectious disease in a human patient).

In an example of the vertebrate or cell of any aspect of the invention,the genome comprises an immunoglobulin light chain locus comprising oneor more human V gene segments and one or more human J gene segmentsupstream of a constant region (eg, a human or a mouse lambda or kappaconstant region).

For rearrangement and expression of heavy chains, the locus comprisescontrol elements, such as an Eμ and Sμ between the J gene segment(s) andthe constant region as is known by the skilled person. In one example, amouse Eμ and Sμ is included in the heavy chain locus between the JHrepertoire and the constant region (ie, in 5′ to 3′ order the locuscomprises the JH gene segment(s), Eμ and Sμ and constant region). In anexample, the Eμ and Sμ are Eμ and Sμ of a mouse 129-derived genome (eg,a 129Sv-derived genome, eg, 129Sv/EV (such as 129S7Sv/Ev (such as fromAB2.1 or AB2.2 cells obtainable from Baylor College of Medicine, Texas,USA) or 129S6Sv/Ev))); in another example, the Eμ and Sμ are Eμ and Sμof a mouse C57BL/6-derived genome. In this respect, the locus can beconstructed in the IgH locus of the genome of a cell selected fromAB2.1, AB2.2, VGF1, CJ7 and FH14. VGF1 cells were established anddescribed in Auerbach W, Dunmore J H, Fairchild-Huntress V, et al;Establishment and chimera analysis of 129/SvEv- and C57BL/6-derivedmouse embryonic stem cell lines. Biotechniques 2000; 29:1024-8, 30, 32,incorporated herein by reference.

Additionally or alternatively, the constant region (or at least a Cμ; orCμ and gamma constant regions thereof) is a constant region (or Cμ; orCμ and gamma constant regions thereof) is of a genome described in theparagraph immediately above.

A suitable source of human DNA sequences or gene segments will bereadily apparent to the skilled person. For example, it is possible tocollect a DNA sample from a consenting human donor (eg, a cheek swabsample as per the Example herein) from which can be obtained suitableDNA sequences for use in constructing a locus of the invention. Othersources of human DNA are commercially available, as will be known to theskilled person. Alternatively, the skilled person is able to constructgene segment sequence by referring to one or more databases of human Iggene segment sequences disclosed herein.

In an example, the genome comprises all or some of the following humanVH gene segments

IGHV6-1 IGHV3-7 IGHV1-8 IGHV3-9 IGHV3-11 IGHV3-13 IGHV1-18 IGHV3-30IGHV4-31 IGHV4-39 IGHV4-59

Optionally also (i) and/or (ii)

(i) IGHV1-2 IGHV2-5 and IGHV3-21 (ii) IGHV1-2 IGHV2-5 IGHV3-21 IGHV1-24(ii) IGHV1-2*04 IGHV2-5*10 and IGHV3-21*03 (iv) IGHV1-2*02 IGHV2-5*01IGHV3-21*01 and IGHV1-24*01

For example, the genome comprises all or some of the following human VHgene segment variants

IGHV6-1*01 IGHV3-7*01 IGHV1-8*01 IGHV3-9*01 IGHV3-11*01 IGHV3-13*01IGHV1-18*01 IGHV3-30*18 IGHV4-31*03 IGHV4-39*01 and IGHV4-59*01;

Optionally also (iii) or (iv)

For example, the genome comprises all or some of the following human JHgene segment variants

IGHJ2*01 IGHJ3*02 IGHJ4*02 IGHJ5*02 and IGHJ6*02

For example, the genome comprises all or some of the following human Dgene segments

IGHD1-1 IGHD2-2 IGHD3-9 IGHD3-10 IGHD5-12 IGHD6-13 IGHD1-14 IGHD2-15IGHD3-16 IGHD4-17 IGHD6-19 IGHD2-21 IGHD5-24 IGHD1-26 and IGHD7-27and optionally also (v) or (vi)

(v) IGHD3-3 (vi) IGHD3-3 IGHD4-4 IGHD5-5 IGHD6-6 IGHD1-7 IGHD2-8 andIGHD2-8

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claimsand/or the specification may mean “one,” but it is also consistent withthe meaning of “one or more,” “at least one,” and “one or more thanone.” The use of the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or.” Throughoutthis application, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples (Examples 1-3 being prophetic). Example 4 is a workedexample.

EXAMPLES Example 1 Recombineered BAC Vectors to add PolymorphicV-regions to the Mouse Genome

FIG. 1 through 3 depict recombineering methods (see references above)that can be used to introduce polymorphic V-gene regions into genomicDNA. In one embodiment, a genomic fragment from the human heavy chainregion is inserted into a bacterial artificial chromosome (BAC) vectorby standard techniques. Preferably, such a BAC, which can range in sizefrom 20-kb to 200-kb or more, can be isolated from libraries of BACs bystandard techniques including sequence searches of commerciallyavailable libraries or by hybridization to bacterial colonies containingBACs to identify those with a BAC of interest.

A BAC is chosen that has several VH gene segments; in FIG. 1, these aregenerically identified as VH[a] through VH[z] for example. One skilledin the art will readily identify appropriate genomic fragments, forexample, an approximately 120-kb fragment from human VH5-78 throughVH1-68 which includes 5 endogenous active VH gene segments and 7 VHpsuedogenes. Using recombineering techniques, the endogenous VH genesegments can be replaced by polymorphic VH or VL gene segments. In thisexample, two steps are required. The first step replaces the V-regioncoding exon of an endogenous VH gene segment with a positive-negativeselection operon, in this example, an operon encoding an ampicillinresistance gene (Amp) and a streptomycin-sensitizing ribosomal protein(rpsL). Certain strains of bacteria can be selected for the absence ofthe rpsL gene by resistance to streptomycin. Short stretches of DNAhomologous to sequences flanking the endogenous VH gene exon are placed5′ and 3′ of the rpsL-Amp operon. In the presence of appropriaterecombination factors per standard recombineering techniques (seereferences above) recombination between the operon fragment and the BACwill result in replacement of the endogenous VH gene exon with theoperon (FIG. 1a ) which are selected by resistance to ampicillin. Thesecond step uses the same homologous sequences in order to replace theinserted operon with a desired polymorphic VH gene segment. In thisexample, a human VH1-69 gene is inserted (FIGS. 1b and 1c ). Inparticular the *02 allele of VH1-69 is used [ref IMGT and FIG. 5].Successful integrations of the polymorphic VH gene segment are selectedin bacteria that become resistant to streptomycin due to the loss of theoperon, specifically the rpsL portion.

In this example, the two step process as described can be repeated foreach of the endogenous VH gene segments or for as many endogenous genesegments that one wishes to replace with polymorphic V gene segments(FIG. 1d ).

As is apparent, any polymorphic V gene segment can be inserted in thismanner and any endogenous V gene segment can act as a target, includingpseudogenes. V gene segments in each of the heavy chain and two lightchain loci can be replaced using this technique with appropriate genomicfragments available as BAC inserts.

FIG. 2 depicts another method for creating a genomic fragment encodingpolymorphic V gene segments. In this example, polymorphic V genesegments are inserted into a region of genomic DNA devoid of othergenes, control elements or other functions. Such ‘desert’ regions can beselected based on sequence analysis and corresponding DNA fragmentscloned into BACs or identified in existing BAC libraries. Starting withsuch a genomic fragment, recombineering techniques can be used to insertpolymorphic V gene segments at intervals of, for example, 10-kb. In thisexample, a 150-kb genomic fragment might accommodate insertion of up to15 polymorphic V gene segments. Insertion of the segments is a two-stepprocess. The first recombineering step inserts the rpsL-Amp operon at aspecific site. Sequences homologous to a specific site are used to flankthe operon. These are used by the recombineering system to insert theelement specifically into the BAC genomic fragment and positive eventsare selected by resistance to ampicillin (FIG. 2a ). The second stepreplaces the operon in the genomic fragment with a polymorphic V genesegment by a similar recombineering step using the same sequencehomology (FIG. 2b ). In this example, both exons and promoter element ofa polymorphic VH gene segment are inserted, resulting in replacement ofthe rpsL-Amp operon and therefore resistance to streptomycin (FIG. 2c ).

The two step technique for inserting polymorphic V gene segments into aspecific site on the genomic fragment can be repeated multiple timesresulting in a BAC genomic fragment with several polymorphic genesegments, including their promoter elements. It is apparent that theexamples shown in FIGS. 1 and 2 can be combined wherein the techniquefor insertion can be used to add extra polymorphic V gene segments to aBAC genomic fragment as depicted in FIG. 1. One might choose to addthese extra segments to an IG genomic fragment since such a fragmentwould be more amenable to proper IG gene expression once inserted into anon-human mammal's genome. It is known that a genomic fragment can haveelements such as enhancers or elements that contribute to certainchromatin conformations, both important in wild-type gene expression.

FIG. 3 depicts an additional method to create genomic fragments withpolymorphic V gene segments. This method depends upon the efficiencywith which short (around 50 to 150 bases, preferably 100 bases) singlestranded DNA fragments recombine with a homologous sequence usingrecombineering (Nat Rev Genet. 2001 October; 2(10):769-79;Recombineering: a powerful new tool for mouse functional genomics;Copeland N G, Jenkins N A, Court D L). The recombinases used inrecombineering preferentially bind and use such short single-strandedfragments of DNA as a substrate for initiating homologous recombination.The efficiency can be as high as 10-2, that is, a positive event can befound in approximately 100 randomly picked (not selected) clonesresulting from recombineering. A positive event in this exampleoccurring when one or more single nucleotide changes introduced into thesingle-stranded fragment get transferred to the BAC insert containing Vgene segments and surrounding genomic DNA, said nucleotide change orchanges occurring at a homologous sequence on the BAC.

Polymorphic V gene segments can differ from endogenous V gene segmentsby only 1 or 2, or up to 10 or 15 nucleotide changes, for example. Anexample of such nucleotide polymorphisms are depicted in FIG. 5. Shortsingle stranded regions that encompass the polymorphic nucleotidechanges can be chemically synthesized using standard techniques. Theresulting single stranded DNA fragments are introduced into bacteria andvia recombineering techniques approximately 1 in 100 BAC fragments willhave incorporated the polymorphic nucleotides via homologousincorporation of the single stranded fragment (FIG. 3a ). BACs with thedesired nucleotide change can be identified by screening for exampleseveral hundred individual clones by polymerase chain reaction (PCR)amplification and sequencing, both by standard techniques. In theexample, two nucleotide changes will convert a VH1-69*01 gene segmentinto a VH1-69*02 gene segment (FIG. 3b ).

It is clear that this process can be repeated for multiple endogenous Vgene segments contained on a single BAC genomic fragment. In addition,the techniques depicted in FIG. 2 can be used to add additionalpolymorphic V gene segments by insertion into regions between existing Vgene segments. As would be evident to one skilled in the art, acombination of these techniques can be used to create numerousvariations of both polymorphic and endogenous human V gene segments. Andit would be evident that several different genomic fragments withengineered polymorphic V gene segments and endogenous human V genesegments can be combined to create even more variations.

Example 2 Adding Polymorphic V-regions to the Genome using SRMCE ofModified BACs

Modified BACs with polymorphic V gene segments created using the methodsdescribed in Example 1 can be used to alter the genome of non-humanmammals. These alterations can result in an intact IG locus in whichnormal immunoglobin region recombination results in VDJ or VJcombinations which includes the human V gene segments. An example of howsuch an animal can be created is by altering the genome of, for example,mouse embryonic stem (ES) cells using the strategy outlined in FIG. 4.

One technique to integrate modified BACs with polymorphic V genesegments into a genome is sequential recombinase mediated cassetteexchange (SRMCE). The technique is described in WO2011004192 (GenomeResearch Limited), which is incorporated here in its entirety byreference.

SRMCE provides for a locus modified with a ‘landing pad’ inserted at aspecific location. This insertion can either be de novo via homologousrecombination or as a consequence of a previous BAC insertion. In thisexample, the landing pad is inserted in the mouse IGH locus between themost 3′ J gene segment and the Cu gene segment and a previous BACinsertion via SRMCE techniques have resulted in the addition of 5 humanV gene segments and 2 V region pseudogenes. The landing pad has elementsas shown in FIG. 4 that will allow the selection of correct insertion ofa second targeting BAC fragment. The specificity of this insertion isprovided by cre recombinase-mediated exchange between permissive loxsites. A lox site is permissive for recombination only with a compatiblelox site. In this example, the loxP site will only recombine with loxPand lox2272 will only recombine with lox2272. This providesdirectionality to the insertion of the BAC fragment as depicted in FIGS.4b and 4 c.

ES cell clones with correct insertions are selected from a pool ofclones without insertions or with non-productive insertions byresistance to puromycin. Resistance to puromycin results from thejuxtaposition of an active promoter element, PGK, with the puroTK codingregion. Correct insertions are verified by standard techniques includingPCR of junctions, PCR of internal elements, Southern blotting,comparative genomic hybridization (CGH), sequencing and etc. In theexample, correct lox2272-lox2272 and loxP-loxP recombination alsoresults in two intact sets of piggyBac elements that did not exist priorto insertion. An intact piggyBac element is comprised of a set ofinverted repeats which are depicted in the figure by “PB5′” and “PB3′”.An appropriated oriented set of piggyBac elements are the substrate ofpiggyBac transposase which can catalyse recombination between theelements, resulting in deletion of intervening sequences as well as bothelements. The DNA remaining after a piggyBac transposition is leftintact and is lacking any remnant of the piggyBac element. In theexample, ES cell clones with successful piggyBac transposition areselected by loss of the active puroTK element which renders the cellsresistant to the drug FIAU (FIGS. 4c and 4d ).

The final product of the SRMCE method in this example is a IGH locuswith several polymorphic V gene segments inserted along with a set ofendogenous unmodified VH gene segments between sequences of the mousegenome on the 5′ side and the mouse IGH constant region gene segments onthe 3′ side. The polymorphic V gene segments are positioned such thatthey can participate in the recombination events associated with B cellmaturation yielding VDJ gene segments. These gene segments can then betranscribed and spliced to the mouse constant region. Translation ofthese transcripts will result in the production of an antibody heavychain encoded by the polymorphic V gene segment, a human DH genesegment, a human JH gene segment and a mouse constant heavy chain genesegment.

As is well known to those skilled in the art, an ES cell clone can beused to create a line of genetically modified mice via injection of saidcells into a mouse blastocyst embryo, transferring the injected embryoto a suitable recipient and breeding the chimeric offspring that result.The modified gene locus can be propagated through breeding and madeeither heterozygous or homozygous depending on the genetic cross.

It is evident from the structure of the IGH locus provided in thisexample and by knowledge of the mechanisms involved in B cell receptor(BCR) and antibody gene rearrangements that a large set of differentcombinations of polymorphic V gene segments with various DH and JH genesegments will result and these can contribute to a large repertoire offunctional antibody genes in a population of B cells in geneticallymodified animals. In this example, several different human VH1-69polymorphs are incorporated to provide superhuman VH diversity. Thisparticular VH gene segment is known to be prevalent in antibodies thatbind infectious disease pathogens (such as influenza virus) andtherefore the antibody repertoire of a mouse with the geneticmodification of this example would be expected to produce antibodieswith a bias in favour of those that bind infectious disease pathogens.The repertoire, in other words, would have a larger subset of antibodieswith superior affinities for pathogen antigens. Examples of suchpathogens include influenza virus, hepatitis C virus (HCV) and humanimmunodeficiency virus-1 (HIV-1) (see also table above).

Example 3

“Alignment of 13 VH1-69 Alleles”

Building a more diverse antibody repertoire by incorporating additionalV gene segment polymorphs requires availability of polymorphic allelesof V gene segments. One source of such alleles include sequencedatabases. In this example, 13 distinct alleles of the VH1-69 genesegment are provided. These allele sequences and comparisons are drawnfrom the “IMmunoGeneTics” IMGT Information System (www.imgt.com)database. FIG. 5 is a diagram of the alignment of alleles *02 through*13 with the *01 allele. The VH1-69*01 nucleotide and amino acidsequence is provided at the top of the figure. Where the remainingalleles are identical to the *01 allele sequence a dash is insertedbelow the sequence. Nucleotide differences are noted alongside theappropriate allele and if the sequence change results in a proteincoding change, the amino acid change is indicated above the triplet.

FIG. 5 depicts between 1 and 4 amino acid changes for each allele incomparison to the *01 allele. All of the amino acid changes occur in thepart of the heavy chain protein encoding the complementarity determiningregions (CDRs). These regions are responsible for antigen specificityand the affinity of the antibody for the antigen. It is evident thatproviding additional polymorphic CDRs in a repertoire of antibodies willincrease the likelihood of there being an antibody with superior bindingcharacteristics for various antigens. In several reports, it has beenobserved that the VH1-69-encoded variable region of the heavy chain isoften found in antibodies that bind influenza virus, HCV and HIV-1antigens (see table above). Therefore incorporating the polymorphic Vgene segments of this example into a transgenic animal model using themethods of Examples 1 and 2 would likely result in an antibodyrepertoire in said transgenic animal with more antibodies that bind toantigens associated with these and other pathogens. And as is known inthe art, a larger repertoire increases the probability of findingmonoclonal antibodies using, for example, hybridoma technology, thatbind with high affinity and specificity to a desired antigen.

This disclosure therefore describes in these examples a transgenic mousemodel which can be immunized with pathogen or other antigens. Plasma Bcells from such an immunized mouse can be used to make a hybridomalibrary that can be screened for production of antibodies that bind thepathogen antigens. This library will be superior to libraries fromtraditional transgenic mice for finding such antibodies given theaddition of polymorphic VH1-69 gene segments to the IGH locus in saidtransgenic mouse.

These examples are not limiting to the human polymorphic V gene segmentsthat can be chosen or to the methods used to introduce them into ananimal model. The method can be used to construct a transgenic locuswith immunoglobulin D and/or J segments. The V, D, J segments can befrom a plurality of human sources (optionally more than one human ethnicpopulation).

Example 4 Transgenic Mice, B-Cells, Hybridomas, Antibodies & HeavyChains Based on Human JH6*02

A functional human gene segment repertoire (from V_(H)2-26 to J_(H)6,see the IMGT database for the structure of the human IgH locus;

http://www.imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=locus&species=human&group=IGK)was sectored by the inventors to produce two different transgenic heavychain alleles (denoted S2F and S3F) and corresponding mice. Thetransgenic alleles were expressed in the mice and the heavy chainrepertoires were assessed at the RNA transcript level. Deep sequenceanalysis was carried out using Bioinformatics methods to assess V, D andJH gene usage, including in variable domain sequences having a HCDR3length of at least 20 amino acids. Endogenous, mouse variable regiongene segments were inactivated by inversion (as per the method describedin WO2011004192, this disclosure being incorporated herein byreference).

Sequencing of Human Donor DNA Samples: Identification of ConservedJH6*02 Variant

DNA samples from 9 anonymised consenting human donors were obtained bytaking cheek swabs.

The samples were processed and the DNA Samples were extracted follow theprotocol of QIAamp DNA Mini Kit (Cat. No. 51304, Qiagen).

PCR reactions were set up to amplify the JH6 region and PCR productswere sequenced (PCR Oligos sequence: Fwd. 5′-AGGCCAGCAGAGGGTTCCATG-3′(SEQ ID NO: 444), Rev. 5′-GGCTCCCAGATCCTCAAGGCAC-3′ (SEQ ID NO: 445)).

Sequence analysis was carried out by comparing to the JH6 referencesequence from IMGT annotated database (http://www.imgt.org/), and thisidentified that all 9 donor genomes contained the human JH6*02 variant,with this variant being in the homozygous state in 7 out of the 9donors. The inventors also consulted the genomic sequences publiclyavailable for Jim Watson and Craig Venter at Ensembl human genomedatabase [http://www.ensembl.org/]. These too contained the human JH6*02variant. This confirmed to the inventors that human JH6*02 is a common,conserved variant in humans, and thus a good candidate for constructionof a transgenic IgH locus as per the invention

Identification of Suitable Human DNA Sequence BACs

A series of human bacterial artificial chromosome (BAC) clones wereidentified from Ensemble (http://www.ensembl.org/index.html) or UCSC(http://genome.ucsc.edu/) human database searches based on gene name(IGH) or location (chromosome 14: 106026574-107346185). Seven human RP11BAC clones were selected, RP11-1065N8 BAC carrying human JH6*02. Intotal, the following BACs were identified as sources of human IgH locusDNA: RP11-1065N8, RP11-659B19, RP11-14117, RP-112H5, RP11-101G24,RP11-12F16 and RP11-47P23.

With a similar approach, different BAC clones (eg, different RP11 cloneIDs or different sources from RP11) or genetically engineered BACs canbe selected for insertion into the mouse IGH locus to provide differentsets of human repertoires in the transgenic mouse.

Construction of Transgenic IgH Loci

Insertion of human heavy gene segments from a 1st IGH BAC (RP11-1065N8)into the IGH locus of mouse AB2.1 ES cells (Baylor College of Medicine)was performed to create a heavy chain allele denoted the S1 allele. Theinserted human sequence corresponds to the sequence of human chromosome14 from position 106494908 to position 106328951 and comprisesfunctional heavy gene segments V_(H)2-5, V_(H)7-4-1, V_(H)4-4, V_(H)1-3,V_(H)1-2, V_(H)6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14,D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24,D6-25, D1-26, D7-27, J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5 and J_(H)6(in 5′ to 3′ order), wherein the JH6 was chosen to be the human JH6*02variant. The insertion was made between positions 114666435 and114666436 on mouse chromosome 12, which is upstream of the mouse Cμregion. The mouse V_(H), D and J_(H) gene segments were retained in thelocus, immediately upstream of (5′ of) the inserted human heavy chainDNA.

A second allele, S2 was constructed in which more human functional V_(H)gene segments were inserted upstream (5′) of the 5′-most V_(H) insertedin the S1 allele by the sequential insertion of human DNA from a secondBAC (BAC2). The inserted human sequence from BAC2 corresponds to thesequence of human chromosome 14 from position 106601551 to position106494909 and comprises functional heavy chain gene segments V_(H)3-13,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7. The mouse V_(H), D and J_(H)gene segments were retained in the locus, immediately upstream of (5′of) the inserted human heavy chain DNA. In a subsequent step, these wereinverted to inactivate them, thereby producing S2F mice in which onlythe human heavy chain variable region gene segments are active.

A third allele, S3 was constructed in which more human functional V_(H)gene segments were inserted upstream (5′) of the 5′-most V_(H) insertedin the S2 allele by the sequential insertion of human DNA from a thirdBAC (BAC3). The inserted sequence corresponds to the sequence of humanchromosome 14 from position 106759988 to position 106609301, andcomprises functional heavy chain gene segments, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, and V_(H)3-15. The mouseV_(H), D and J_(H) gene segments were retained in the locus, immediatelyupstream of (5′ of) the inserted human heavy chain DNA. In a subsequentstep, these were inverted to inactivate them, thereby producing S3F micein which only the human heavy chain variable region gene segments areactive.

Mice bearing either the S2F or S3F insertion into an endogenous heavychain locus were generated from the ES cells using standard procedures.The other endogenous heavy chain locus was inactivated in the mice byinsertion of an inactivating sequence comprising neo^(R) into the mouseJ_(H)-Cμ intron (to produce the “HA” allele).

Specifically, the following alleles were included:—

VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3-7*01, VH3-9*01,VH3-11*01, VH3-13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 andVH7-4-1*01 D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01,D6-13*01 and D6-19*01 Immunisation Procedure

Transgenic mice of the S2F or S3F genotype were primed with 20-40 ugrecombinant proteins obtained commercially or produced in house withAntigen 1 (OVA (Sigma A7641); Antigen 2 (a human infectious diseasepathogen antigen) and Antigen 3 (a human antigen) via the ip route incomplete Freunds adjuvant (Sigma F 5881) and 10 ug/animal CpG (CpGoligo; Invivogen, San Diego, Calif., USA) and then boosted twice inabout two weekly intervals with about half the amount of antigen inincomplete Freunds adjuvant (Sigma F 5506) and 10 ug/animal CpG. Finalboosts were administered two weeks later iv without any adjuvant andcontained 5-10 ug protein in PBS.

Hybridoma Fusion Procedure

Spleens were taken 3 days after the final boost and spleenocytes weretreated with CpG (25 μm final concentration) for and left until thefollowing day. Cells were then fused with SP0/2 Ag14 myeloma cells (HPACultures Cat No 85072401) using a BTX ECM2001 electrofusion instrument.Fused cells were left to recover for 20 minutes then seeded in a T75flask until next morning. Then the cells were spun down and plated outby dilution series on 96-well culture plates and left for about 10 daysbefore screening. Media was changed 1-3 times during this period.

Screening

Culture supernatants of the hybridoma wells above were screened usinghomogenious time resolved fluorescence assay (htrf) using Europiumcryptate labelled anti-mouse IgG (Cisbio anti-mouse Ig EuropiumCryptate) and a biotin tagged target antigen with a commerciallyavailable streptavidin conjucated donor (Cisbio; streptavidingconjugated D2) or by IgG-specific 384 well ELISA. Positive wellsidentified by htrf were scaled to 24-well plates or immediatelycounterscreened using an IgG-specific detection ELISA method. Positivesidentified by primary ELISA screen were immediately expanded to 24-wellplates. Once cultures were expanded to 24-well stage and reachedconfluency, supernatants were re-tested using htrf or IgG-specific ELISAto confirm binding to target antigen. Supernatant of such confirmedcultures were then also analysed by surface plasmon resonance using aBioRad ProteOn XPR36 instrument. For this, antibody expressed in thehybridoma cultures was captured on a biosensor GLM chip (BioRad 176-512)which had an anti-mouse IgG (GE Healthcare BR-1008-38)) covalentlycoupled the biosensor chip surface. The antigen was then used as theanalyte and passed over the captured hybridoma antibody surface. ForAntigen 2 and Antigen 3, concentrations of 256 nM, 64 nM, 16 nM, 4 nMand 1 nM were typically used, for Antigen 1, concentrations of 1028 nM,256 nM, 64 nM, 16 nM and 4 nM were typically used, binding curves weredouble referenced using a 0 nM injection (i.e. buffer alone). Kineticsand overall affinities were determined using the 1:1 model inherent tothe BioRad ProteOn XPR36 analysis software.

Any clones with confirmed binding activity were used for preparing totalRNA and followed by PCR to recover the heavy chain variable regionsequences. Standard 5′-RACE was carried out to analyse RNA transcriptsfrom the transgenic heavy chain loci in the S2F and S3F mice.Additionally, deep sequence analysis of almost 2000 sequences producedby the mice was carried out.

Bionformatics Analysis

Sequences for analysis were obtained from two different methods:

-   -   The first is from RNA extracted from the spleen: first cDNA        strand was synthesized using an oligo based on the Cmu region of        the mouse IGH locus as a PCR template. PCR was performed using        this oligo with an oligo dT-anchor primer. Then PCR product was        cloned into pDrive vector (Qiagen) and then sequenced.    -   The second is from hybridomas generated through electro-fusion:        total RNA was extracted from hybridoma lines of interest using        standard Trizol methods and frozen at −80° C. for long term        storage. cDNA was generated from 100 ng total RNA using standard        Superscript III reverse transcriptase and a gene-specific        reverse primer binding to all mouse IgG isotypes for heavy chain        and a mouse kappa constant region primer for the light chain        amplification. 2-3 ul of cDNA were then used as template in a        PCR reaction using Pfu DNA polymerase and a panel of degenerate        forward primers annealing to the leader sequence of the human        immunoglobulin variable domain as well as one mouse pan-IgG        reverse primer. PCR products were run out of a 1% agarose gel        and bands of approximately 350-450 basepairs extracted and        purified. DNA was then sequenced.

The sequences from the first method can either be from IgM from Naïvemice or IgG from immunised mice. The samples from the second method areall from IgG from immunised mice, and specific to the immunisingantigen. Almost 2000 sequences were analysed.

The sequences were obtained as a pair of forward and reverse reads.These were first trimmed to remove low-quality base calls from the endsof the reads (trimmed from both ends until a 19 nucleotide window had anaverage quality score of 25 or more). The reads were combined togetherby taking the reverse complement of the reverse read, and aligning itagainst the forward read. The alignment scoring was 5 for a match, −4for a mismatch, a gap open penalty of 10 and a gap extension penaltyof 1. A consensus sequence was then produced by stepping through thealignment and comparing bases. When there was a disagreement the basewith the highest quality value from sequencing was used.

The BLAST+ (Basic Local Alignment Search Tool) (Camacho C., CoulourisG., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T. L. (2008)“BLAST+: architecture and applications.” BMC Bioinformatics 10:421http://www.ncbi.nlm.nih.gov/pubmed/20003500) program ‘blastn’ was thenused to find the germline J and V segments used in each sequence. Awordsize of 30 was used for V matching, and 15 for J matching. Thedatabase searched against was constructed from the NGS sequencing of theBACs which were used to generate the Kymouse.

If a sequence matched both a V and a J segment, the sequence between thetwo was then compared to a database of germline D segments in the mouseusing ‘blastn’ with a wordsize of 4 and the options ‘blastn-short’ and‘ungapped’. This was used to assign a D segment, if possible. The CDR3was identified by searching for the conserved “TATTACTGT” sequence inthe V segment, and the “CTGGGG” in the J segment. If these motifs werenot found, then up to 4 mismatches were allowed. The IMGT definition ofCDR3 was used, so the CDR3 length is calculated from after the “TGT” inthe V to before the “TGG” in the J. Sequences with an out of framejunction (those which do not have a CDR3 nucleotide length divisible by3) or which contained a stop codon (“TAA”, “TAG” or “TGA”) wereexcluded.

The identity of the matching V, J and D segments as well as the CDR3length from this assignment were then saved as a table for downstreamanalysis. The ratio of IGHJ6*02 used increased from the naïve toimmunised mice, as well as being enriched in the sub-population ofsequences with a long HCDR3 (defined as consisting of 20 or more aminoacids):

All HCDR3 > 20 Total Total JH6*02% Count JH6*02% Count % HCDR3 > 20Naïve 22.31% 1340 91.11% 45 3.36% Immunised 37.50% 256 66.67% 9 3.52%Hybridoma 36.13% 119 63.64% 11 9.24%

This shows that the JH6*02 gene segment is selected for by immunisation,as the proportion of JH6*02 usage increases after immunisation. JH6*02is also used in the majority of antibodies with a long HCDR3 length,which is desirable for targets which are specifically bound by longHCDR3 length antibodies.

Additionally, the analysis revealed that certain VH and D gene segmentsfrequently yielded HCDR3s of long length (in all of naïve, immunised andantigen-specific repertoires of heavy chains). See Table 2.

TABLE 2 Long HCDR3s from Naïve Repertoires Average CDR3 V Length CountIGHV1-2*02 21 3 IGHV1-18*01 21 5 IGHV3-7*01 22 3 IGHV6-1*01 21 5IGHV3-9*01 20 2 IGHV2-5*10 20 1 IGHV7-4-1*01 21 3 IGHV1-3*01 21 5IGHV4-4*02 20 3 IGHV3-13*01 22 1 IGHV3-23*04 20 1 IGHV1-8*01 21 10IGHV3-21*03 23 3 Average CDR3 D Length Count IGHD2-2*02 20 1 IGHD3-9*0121 13 IGHD3-10*01 21 26 IGHD6-13*01 20 1 IGHD4-17*01 22 2 IGHD6-19*01 231 IGHD3-22*01 20 1 CDR3 Length (All Naïve) Count 20 23 21 10 22 7 23 324 1 26 1 Long HCDR3s from Immunised Repertoires Average CDR3 V LengthCount IGHV4-4*02 20 1 IGHV3-11*01 23 2 IGHV3-7*01 21 6 Average DCDR3Length Count IGHD2-2*02 22 2 IGHD3-10*01 22 5 IGHD6-19*01 20 1IGHD1-26*01 20 1 CDR3 Length (All Immunised) Count 20 4 21 1 22 2 24 125 1 Long HCDR3s from Antigen-Specific Repertoires Average CDR3 V LengthCount IGHV4-4*02 20 2 IGHV1-3*01 21 3 IGHV3-11*01 21 1 IGHV3-7*01 22 1IGHV1-8*01 22 2 IGHV3-20*d01 22 1 IGHV3-9*01 20 1 Average CDR3 D LengthCount IGHD2-2*02 22 1 IGHD3-9*01 21 1 IGHD3-10*01 21 9 CDR3 Length (AllAntigen- Specific) Count 20 4 22 2 21 4 24 1

What is claimed is:
 1. A method of producing an antigen specificantibody or antigen binding fragment thereof, said antibody comprising ahuman immunoglobulin heavy (IgH) chain, wherein said human IgH chaincomprises a human IgH chain variable region and a human IgH chainconstant region, and said fragment comprising a human IgH chain variableregion, the method comprising: expressing the antibody or antigenbinding fragment thereof from a host cell comprising nucleic acidencoding said human IgH chain variable region and said human IgH chainconstant region of said antibody, or comprising nucleic acid encodingsaid human IgH chain variable region of said fragment, wherein saidhuman IgH chain variable region of said antigen-specific antibody orantigen binding fragment is of a B cell, or a hybridoma thereof, of atransgenic mouse contacted with said antigen; wherein said transgenicmouse comprises a germline comprising: (i) an immunoglobulin heavy (IgH)chain locus comprising a plurality of human VH, one or more human D andone or more human JH gene segments at an endogenous locus upstream ofand operably linked to a constant region; wherein said plurality ofhuman VH gene segments is selected from at least two of the groupconsisting of: IGHV3-7*01, IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01,IGHV4-4*02, IGHV3-13*01, IGHV3-23*04 and VH3-20*d01, and where one ormore human D gene segments is selected from the group consisting of:IGHD2-2*01, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01,IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01, wherein each of said selectedhuman VH gene segments of said plurality of human VH gene segments iscapable of joining with a human D gene segment and a human JH genesegment to encode a variable region, wherein each of said selected humanVH gene segments of said plurality of human VH gene segments is capableof joining with a human D gene segment and a human JH segment to encodea variable region comprising a HCDR3 of 20 or more amino acids in lengthin said mouse; (ii) an immunoglobulin kappa light (Igκ) chain locuscomprising one or more human Igκ variable (Vκ) gene segments and one ormore human Igκ joining (Jκ) gene segments upstream of and operativelylinked to a constant region; wherein said one or more Vκ gene segmentsis selected from the group consisting of a VκI gene family member,κ1-15A (KL012), a VκII family member, a VκIII family member, VκII A2,VκII A2a and Vκ A27 (Humkv325), such that said transgenic mouse iscapable, upon contact with an antigen, of producing a plurality ofantibodies specific for said antigen comprising an immunoglobulin heavychain comprising a human VH region and a C region; wherein saidplurality of antibodies specific for said antigen comprises a human VHregion having an HCDR-3 length of at least 20 amino acids.
 2. The methodof claim 1, wherein said one or more human D gene segments comprisesIGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01,IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01.
 3. The method of claim 1,wherein the C region of the IgH locus is a mouse C region.
 4. The methodof claim 1, wherein said B cells of said transgenic mouse contacted withantigen are isolated.
 5. The method of claim 4, comprising the step ofisolating antigen-specific antibody from said isolated B cells.
 6. Themethod of claim 4, further comprising the step of isolating from said Bcells nucleic acid encoding said human VH region and said mouse C regionof said chimeric immunoglobulin heavy chain.
 7. The method of claim 6,further comprising the step of replacing in said nucleic acid encodingsaid human VH region and said mouse C region of said chimericimmunoglobulin heavy chain polypeptide, the portion encoding said mouseC region with nucleic acid encoding a human C region, thereby forming anucleic acid encoding said human VH region and said human C region. 8.The method of claim 4, further comprising isolating an antibody thatbinds said antigen, comprising (i) immortalizing said isolated B cellsexpressing said antigen-specific antibody or progeny thereof, optionallyproducing hybridomas therefrom; and (ii) isolating an antibody expressedby the immortalized B cells of (i).
 9. The method of claim 1, whereinbefore said contact with antigen, said mouse is naïve.
 10. The method ofclaim 1, wherein said antibody is an IgG-type antibody.
 11. The methodof claim 1, wherein said antigen is an antigen of an infectious diseasepathogen.
 12. The method of claim 1, wherein the antigen is of a viralpathogen.
 13. The method of claim 1, wherein expression of mouse Igheavy chain comprising a mouse IgH variable region and a mouse IgHconstant region is inactive in said mouse.
 14. A method of producing anantigen specific antibody or antigen binding fragment thereof, saidantibody comprising a human immunoglobulin heavy (IgH) chain, whereinsaid human IgH chain comprises a human IgH chain variable region and ahuman IgH chain constant region, and said fragment comprising a humanIgH chain variable region, the method comprising expressing the antibodyor antigen binding fragment thereof from a host cell comprising nucleicacid encoding said human IgH chain variable region and said human IgHchain constant region of said antibody, or comprising nucleic acidencoding said human IgH chain variable region of said fragment, whereinsaid human IgH chain variable region of said antigen-specific antibodyor antigen binding fragment is of a B cell, or a hybridoma thereof, of atransgenic mouse contacted with said antigen; wherein said transgenicmouse comprises a germline comprising: (i) wherein said immunoglobulinheavy (IgH) chain locus comprising a plurality of human VH, one or morehuman D and one or more human JH gene segments at an endogenous locusupstream of and operably linked to a constant region; whereinIGHV3-7*01, IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01, IGHV4-4*02,IGHV3-13*01, IGHV3-23*04, and VH3-20*d01, and where one or more human Dgene segments is selected from the group consisting of: IGHD2-2*01,IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01,IGHD3-22*01 and IGHD1-26*01, wherein said plurality of human VH genesegments comprise human VH gene segments capable of joining with a humanD gene segment and a human JH gene segment to encode a variable region,wherein said plurality of human VH gene segments comprise human VH genesegments capable of joining with a human D gene segment and a human JHsegment to encode a variable region comprising a HCDR3 of 20 or moreamino acids in length in said mouse; (ii) an immunoglobulin kappa light(Igκ) chain locus comprising one or more human Igκ variable (Vκ) genesegments and one or more human Igκ joining (Jκ) gene segments upstreamof and operatively linked to a constant region; wherein said one or moreVκ gene segments is selected from the group consisting of a VκI genefamily member, κ1-15A (KL012), a VκII family member, a VκIII familymember, VκII A2, VκII A2a and Vκ A27 (Humkv325), such that saidtransgenic mouse is capable, upon stimulation with an antigen, ofproducing a plurality of antibodies specific for said antigen comprisingan immunoglobulin heavy chain comprising a human VH region and a Cregion; wherein said plurality of antibodies specific for said antigencomprises a human VH region having an HCDR-3 length of at least 20 aminoacids.
 15. The method of claim 14, wherein said one or more human D genesegments comprises IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01,IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01.
 16. The method ofclaim 14, wherein the C region of the IgH locus is a mouse C region. 17.The method of claim 14, wherein said B cells of said transgenic mousecontacted with antigen are isolated.
 18. The method of claim 17, furthercomprising the step of isolating antigen-specific antibody from saidisolated B cells.
 19. The method of claim 17, further comprising thestep of isolating from said B cells nucleic acid encoding said human VHregion and said mouse C region of said chimeric immunoglobulin heavychain polypeptide.
 20. The method of claim 19, further comprising thestep of replacing in said nucleic acid encoding said human VH region andsaid mouse C region of said chimeric immunoglobulin heavy chainpolypeptide, the portion encoding said mouse C region with nucleic acidencoding a human C region, thereby forming a nucleic acid encoding saidhuman VH region and said human C region.
 21. The method of claim 17,further comprising isolating an antibody that binds said antigen,comprising (i) immortalizing said isolated B cells expressing saidantigen-specific antibody or progeny thereof, optionally producinghybridomas therefrom; and (ii) isolating an antibody expressed by theimmortalized B cells of (i).
 22. The method of claim 14, wherein beforesaid contact with antigen, said mouse is naïve.
 23. The method of claim14, wherein said antibody is an IgG-type antibody.
 24. The method ofclaim 14, wherein said antigen is an antigen of an infectious diseasepathogen.
 25. The method of claim 14, wherein the antigen is of a viralpathogen.
 26. The method of claim 14, wherein expression of mouse Igheavy chain comprising a mouse IgH variable region and a mouse IgHconstant region is inactive in said mouse.